Methods compositions and kits for imaging cells and tissues using nanoparticles and spatial frequency heterodyne imaging

ABSTRACT

Methods, compositions, systems, devices and kits are provided herein for preparing and using a nanoparticle composition and spatial frequency heterodyne imaging for visualizing cells or tissues. In various embodiments, the nanoparticle composition includes at least one of: a nanoparticle, a polymer layer, and a binding agent, such that the polymer layer coats the nanoparticle and is for example a polyethylene glycol, a polyelectrolyte, an anionic polymer, or a cationic polymer, and such that the binding agent that specifically binds the cells or the tissue. Methods, compositions, systems, devices and kits are provided for identifying potential therapeutic agents in a model using the nanoparticle composition and spatial frequency heterodyne imaging.

RELATED APPLICATIONS

The present application claims the benefit of U.S. provisionalapplication Ser. No. 61/544,419 filed Oct. 7, 2011 and 61/546,484 filedOct. 12, 2011 in the U.S. Patent and Trademark Office, and which areincorporated herein by reference in their entireties.

GOVERNMENT FUNDING

A portion of this work was supported by U.S. Department of Energy grantDE-FG02-08ER15937, U.S. Department of Education GAANN award P200A090076,and the National Institutes of Health grant CA123544. The government hascertain rights in this invention.

TECHNICAL FIELD

Systems, compositions, methods and kits are provided using nanoparticlecompositions and spatial frequency heterodyne imaging for visualizingand detecting cells or a tissue such as a tumor, for identifying apotential therapeutic agent for treating a disease condition, and forpreparing the nanoparticle compositions.

BACKGROUND

Hepatocellular carcinoma (HCC) is the most common form of liver cancerin adults, accounting for approximately three of every four cancers inthe liver (El-Sarag et al. 1999, New England Journal of Medicine 340:745-750). The American Cancer Society estimates that more than 24,000new cases of primary liver cancer develop each year in the UnitedStates, of which approximately 19,000 result in death. HCC is common indeveloping countries, particularly in sub-Saharan Africa and SoutheastAsia (Trevisani et al. 2008 Carcinogenesis 29: 1299-1305; and O'Brien2004 Cancer Journal 10: 67-73).

More than 500,000 people are diagnosed with HCC each year worldwide(Trevisani et al. 2008 Carcinogenesis 29: 1299-1305; and Bruix et al.2006 Oncogene 25: 3848-3856). HCC is difficult to diagnose in itsearliest stages because there are currently no screening testsavailable, and HCC generally becomes symptomatic when the tumor isapproximately 4.5 centimeters to eight centimeters in diameter(Trevisani et al. 2008 Carcinogenesis 29: 1299-1305; and Colombo 1992Hepatol 15: 225-236).

Detection by ultrasound and imaging by computed tomography (CT) scans ormagnetic resonance imaging (MRI) lack ability to definitively andreproducibly diagnose early stage cancers, particularly small HCC tumors(Bruix et al. 2006 Oncogene 25: 3848-3856; Hain et al. 2004 CancerJournal 10: 121-127; Okuda 2000 J. Hepatol 32: 225-237; and Sheu et al.1985 S. Cancer 56: 660-666). Misdiagnosis of HCC yielding false positiveor false negative results is common from these imaging techniques, andthe American Cancer Society estimates that HCC patients have a five-yearsurvival rate of just 10%.

New techniques for imaging and early diagnosis of HCC and other cancersare needed to improve prognosis of cancers such as HCC.

SUMMARY

An aspect of the invention provides a method of imaging cells or atissue, the method including: contacting a sample of the cells or thetissue with a nanoparticle composition containing at least one selectedfrom the group of: a nanoparticle, a polymer layer coating thenanoparticle, and a binding agent that specifically binds a molecularspecies; irradiating the sample with an X-ray beam; and, detecting byX-ray scatter imaging the nanoparticle in the cells or the tissue. Invarious embodiments, the polymer layer is composed of at least one of: apolyethylene glycol, a polyelectrolyte, an anionic polymer, and acationic polymer. The polyelectrolyte in various embodiments includes aprotein, organic acid, or a polysaccharide; for example thepolyelectrolyte is a poly(acrylic acid) or a poly(allylaminehydrochloride).

The method in various embodiments further includes prior to contacting,constructing the nanoparticle with at least one of: a metal, a metaloxide, an inorganic material, an alloy, and an organic material. In arelated embodiment, the method further includes prior to contacting,constructing the nanoparticle with a MRI agent, a positive contrastagent, or a negative contrast agent. For example, the MRI agent includesan oil, a metal (e.g., iron and magnesium) sulfate, a metal chloride, ora metal ammonium citrate.

The method in various embodiments further includes prior to contacting,constructing the nanoparticle with at least one of: silver, copper,gold, cadmium, zinc, nickel, palladium, platinum, rhodium, platinum,manganese, gadolinium, dysprosium, tantalum, titanium, and iron. Forexample, the nanoparticle includes gadolinium-diethylene triaminepentaacetic acid (DTPA).

In various embodiments, the nanoparticles comprise a metallic core or ametallic shell. In various embodiments, the shell electron density isgreater than the core electron density. Alternatively, in certainembodiments the core electron density is greater than the shell electrondensity. In various embodiments, the nanoparticles are non-toxic orbiocompatible.

In an embodiment of the method, constructing the nanoparticle involvesproducing the nanoparticle to have an average diameter of at least aboutfive nanometers (nm). In various embodiments of the method, thenanoparticle has a diameter of at least about: two nm, five nm, ten nm,20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or at leastabout 100 nm. In an alternative embodiment of the method, thenanoparticle is less than about 100 nm in diameter. In an embodiment ofthe method, constructing the nanoparticle includes engineering aplurality of nanoparticles having an average diameter greater than aboutfive nm and less than about 100 nm.

In various embodiments of the method, irradiating the sample includeslocating or inserting an absorption grid adjacent to the sample betweenan X-ray source and a detector. In certain embodiments, the methodinvolves placing the absorption grid millimeters, centimeters or metersin the vicinity of or adjacent to the sample. The method in certainembodiments involves placing the absorption grid between an x-ray sourceand the sample, or alternatively in between the sample and the detector.

Detecting the presence of the nanoparticles in various embodiments ofthe method involves spatial frequency heterodyne imaging, spatialharmonic imaging. The terms, “spatial frequency heterodyne imaging” and“spatial harmonic imaging” are used interchangeably herein. In variousembodiments, the spatial frequency heterodyne imaging involvesperforming a Fourier transformation of x-ray scatter images obtained bythe detector. For example, the detector includes a light sensor such asa camera or a charge-coupled device.

In various embodiments of the method, the binding agent attached orconjugated to the nanoparticle includes at least one molecule selectedfrom the group of: a drug, a protein, a carbohydrate, and a nucleotidesequence. In various embodiments of the method, the protein is anantibody selected from the group of: a monoclonal antibody; a polyclonalantibody; a single-chain antibody (scFv); a recombinant heavy-chain-onlyantibody (VHH); an Fv; a Fab; a Fab′; and a F(ab′)₂. In relatedembodiments of the method, the antibody (e.g., a monoclonal antibody anda polyclonal antibody) specifically binds a tumor antigen selected fromthe group of: aspartyl (asparaginyl)-β-hydroxylase, alpha-fetoprotein,carcinoembryonic antigen (CA), CA-125, mucin 1, epithelial tumorantigen, tyrosinase, melanoma-associated antigen, tumor protein 53,human chorionic gonadotropin, vimentin, CD34, desmin, prostate specificantigen, and glial fibrillary acidic protein. For example, themonoclonal antibody used in the method includes all or a portion of FB50antibody (Wands et al. U.S. Pat. No. 6,797,696 issued Sep. 28, 2004), orSF25 antibody (Wands et al. U.S. Pat. No. 5,212,085 issued May 18,1993).

In a related embodiment, the binding agent binds to or targets geneticmaterial (e.g., a nucleotide sequence) in the cell. In variousembodiments, the genetic material includes a DNA or an RNA, such thatthe RNA is selected from: mRNA, tRNA, rRNA, siRNA, RNAi, miRNA, anddsRNA, or a portion thereof. In certain embodiments, the binding agentbinds to a cell surface receptor or to an intracellular receptor.

In various embodiments, the method images the tissue that contains aplurality of cells selected from at least one of the group of:cancerous, non-cancerous, epithelial, hematopoietic, stem, spleen,kidney, pancreas, prostate, liver, neuron, breast, glial, muscle, sperm,heart, lung, ocular, brain, bone marrow, fetal, blood, leukocyte, andlymphocyte. For example, the tissue is a sample obtained from a subjectfor example the tissue is a portion of an organ (e.g., a liver, heart,brain, and stomach).

In certain embodiments of the method, the binding agent attached orconjugated to the nanoparticle binds to an antigen, or a nucleotidesequence that encodes the antigen. For example, the antigen is a cancerantigen or a tumor antigen. In various embodiments, the method furtherincludes diagnosing or prognosing a disease condition in the subject.

In various embodiments, the method further includes detecting or imaginga tumor in the cells or the tissue, wherein the tumor is selected fromthe group consisting of: melanoma; colon carcinoma; pancreatic;lymphoma; glioma; lung; esophagus; mammary; prostate; head; neck;ovarian; stomach; kidney; liver; and hepatocellular carcinoma.

In various embodiments, the method further includes administering atherapeutic agent to the cells, the tissue, or to the subject. Invarious embodiments, the therapeutic agent is at least one of: anantibiotic, an anti-viral, an anti-cancer, an anti-tumor, ananti-proliferative, and an anti-inflammatory.

An aspect of the invention provides a method of identifying in a modelsystem a potential therapeutic agent for treating or preventing adisease condition, the method including: contacting a first sample and asecond sample of cells or tissue having the disease condition with acomposition containing: a nanoparticle and at least one of a polymerlayer coating the nanoparticle and a binding agent that specificallybinds the disease agent; contacting the second sample with the potentialtherapeutic agent; and, measuring a presence or an amount of a marker inthe first sample and the second sample, such that the marker ischaracteristic of the disease condition, such that a greater amount ofthe marker in the first sample compared to that in the second sample isa measure of treatment and protection by the potential therapeuticagent, thereby identifying the potential therapeutic agent for treatingor preventing the disease condition. In various embodiments of themethod, the composition includes a plurality of nanoparticles.

Detecting the presence of the marker in the first sample and the secondsample includes in various embodiments of the method measuring ordetecting the nanoparticle using X-ray scatter imaging or spatialfrequency heterodyne imaging.

In various embodiments, prior to contacting, the method further includesconstructing the nanoparticle or a plurality of nanoparticles comprisingat least one material selected from the group of: a metal, a metaloxide, a MRI agent, and a combination thereof.

In various embodiments, constructing the nanoparticles involves alayer-by-layer coating of one or more polymers on the nanoparticles. Forexample, at least one of the polymers is selected from: a polyethyleneglycol, a polyelectrolyte, an anionic polymer, and a cationic polymer.

In various embodiments of the method, constructing the nanoparticleincludes forming a shell or core of the nanoparticle with at least onefrom the group of: silver, copper, gold, cadmium, zinc, nickel,palladium, platinum, rhodium, platinum, manganese, gadolinium,dysprosium, tantalum, titanium, and iron. In a related embodiment of themethod, constructing the nanoparticle involves vapor-phase synthesis.

In certain embodiments of the method, the binding agent includes atleast one molecule selected from the group of: a drug, a protein, acarbohydrate, and a nucleotide sequence.

In various embodiments of the method, the disease condition isassociated with or produced by at least one from the group of: a virus,a tumor, a cancer, a fungus, a bacterium, a parasite, a pathogenicmolecule, and a protein. For example, the cancer is a carcinoma such asa hepatoma or a melanoma.

Prior to contacting, the method in various embodiments further includesconstructing the nanoparticle by attaching or conjugating the bindingagent to an external surface of the nanoparticle, such that the bindingagent comprises at least one selected from the group of: a drug, aprotein, a carbohydrate, and a nucleotide sequence. The protein invarious embodiments is an antibody selected from the group of: amonoclonal antibody; a polyclonal antibody; a single-chain antibody(scFv); a recombinant heavy-chain-only antibody (VHH); an Fv; a Fab; aFab′; and a F(ab′)₂.

In various embodiments of the method, measuring further includesobserving a localization of the marker or the nanoparticle in the cellsor the tissue of the first sample and the second sample. For example,the nanoparticle (or plurality of nanoparticles) is located in thenucleus or is located in cytoplasm of a cell in the first sample and thesecond sample.

The method in various embodiments further includes spatial frequencyheterodyne imaging the nanoparticles in the cells or the tissue using anabsorption grid and a detector.

An aspect of the invention provides a kit for imaging cells or a tissuein a subject, the kit including: a composition containing a nanoparticleincluding at least one selected from the group of: a polymer layer and abinding agent, such that the composition binds to and/or is phagocytosedby the cells or the tissue; instructions for use, such that theinstructions describe: contacting the cells or the tissue with thecomposition, and imaging the cells and the tissue and detecting X-rayscattering of the composition with a device; and a container.

The nanoparticles in various embodiments of the kit include at least onematerial selected from the group of: a metal, a metal oxide, aninorganic material, an organic material, a MRI agent, and a combinationthereof.

An aspect of the invention provides a composition for imaging cells or atissue, the composition including: a metal nanoparticle having attachedto an external surface of the nanoparticle a polymer layer, and FB50monoclonal antibody that specifically binds an antigen of hepatocellularcarcinoma, such that the polymer layer comprises at least one of: apolyethylene glycol, a polyelectrolyte, an anionic polymer, and acationic polymer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing of methods for layer-by-layer polyelectrolytecoating of gold nanoparticles using an anionic poly(acrylic acid) (PAA),and a cationic poly(allylamine hydrochloride) (PAH). FIG. 1 Gold (Au)nanoparticles were contacted with PAA to produce PAA-encapsulatednanoparticles (AU-PAA) having carboxylic acid functional groups (—COOH)and de-protonated carboxylic acid functional groups (—COO⁻) extendingfrom the nanoparticles. The PAA-encapsulated nanoparticles werecontacted with PAH to form PAA-PAH encapsulated nanoparticles(Au-PAA-PAH). The PAH layer lies outside the PAA layer of PAA-PAHencapsulated nanoparticle resulting in amine functional groups (—NH₂)and protonated amino functional groups (—NH₃ ⁺) extending from thenanoparticle.

FIG. 2 is a drawing of the X-ray imaging system used in examples herein.An X-ray source directs electromagnetic radiation to an absorption gridthat is a periodic structure positioned in the radiation propagationdirection and positioned proximal to a sample and a charged-coupleddevice (CCD). The grid scatters the directed radiation to the samplepositioned in a container or vial. The X-ray radiation passes throughthe grid and the sample respectively, and is then detected using theCCD.

FIG. 3 is a set of visible light images and X-ray scatter images afterFourier transformation of scattered X-radiation of a sample obtained bythe imaging system shown in FIG. 2. A beam of X-radiation was directedthrough an absorption grid and sample in a vial, and was detected usingan CCD. The original image (F) is shown in the lower left of FIG. 3. AFourier transformation was performed resulting in an image in thespatial frequency domain. Different peaks in the spatial frequency imagecontained different information regarding scattering of incidentx-radiation by the sample. Selecting an area around a specific peak inthe convolution and Fourier back-transforming this area returns thelogarithm of the scattered intensities to real space and gives aprocessed image that contains anisotropic information regardingscattering of the incident X-rays by the sample. The area surroundingthe central 0^(th)-order peak (F⁻¹; S0: 0^(th) order; FIG. 3 bottomright) corresponds to the original X-ray absorption image withoutscatter and is used for normalization. The image from the central0^(th)-order peak is subtracted from the higher order images to removeall absorption features. The area around the 1^(st)-order peak (F⁻¹; S1:1^(st) order left; FIG. 3 center bottom) corresponds to scattering inthe x-direction, and therefore gives a processed left 1^(st)-orderscatter image upon Fourier back-transformation and normalization.Similarly, the area around the 1^(st)-order peak immediately above the0^(th)-order peak (FIG. 3 top right box) corresponds to scattering inthe y-direction, and therefore gives a processed upper 1^(st)-orderscatter image upon Fourier back-transfounation and normalization. EachX-ray image yields at least two processed images; one image results fromX-radiation scattered horizontally (F⁻¹; S1: 1^(st) order left; FIG. 3center bottom), and the other image results from X-radiation scatteredvertically (FIG. 3 top right box). Both 1^(st) order images measureidentical scatter signals because of the isotropic scattering of thespherical nanoparticles.

FIG. 4 panels A-D are a set of images showing vials with cell pelletscontaining approximately 10⁷ FOCUS cells (Friendship of China and UnitedStates; a human hepatocellular carcinoma cell line; He, L. et al. 1984In Vitro 20(6): 493-504) labeled with PAA-PAH coated gold nanoparticleshaving a diameter of 10 nm (right vial in each panel) or 50 nm (leftvial in each panel). Control cells were not contacted with goldnanoparticles (center vial in each panel). Top boxes and bottom boxes ineach of FIG. 4 panels B-D outline areas selected for intensity profilesof the supernatant and pellet, respectively. Data show improvedsensitivity and clarity for spatial harmonic images of coatednanoparticles compared to absorbance images.

FIG. 4 panel A is a camera photograph of FOCUS cell pellets under cellculture medium. Pellets of cells contacted with ten nm PAA-PAH coatedgold nanoparticles (FIG. 4 panel A left vial) and with 50 nm PAA-PAHcoated gold nanoparticles (FIG. 4 panel A right vial) are visibly darkerin appearance that control pellets of cells not contacted with the goldnanoparticles (FIG. 4 panel A center vial).

FIG. 4 panel B is an absorption image of the vials containing FOCUS cellpellets contacted with ten nm PAA-PAH coated gold particles (rightimage), contacted with 50 nm PAA-PAH coated gold particles (left image),or control pellets of cells not contacted with the gold nanoparticles(center image).

FIG. 4 panel C is an left 1st-order processed image of the vialscontaining FOCUS cell pellets contacted with ten nm PAA-PAH coated goldparticles (right image), contacted with 50 nm PAA-PAH coated goldparticles (left image), or control pellets of cells not contacted withthe gold nanoparticles (center image).

FIG. 4 panel D is an upper 1st-order processed image of the vialscontaining FOCUS cell pellets contacted with ten nm PAA-PAH coated goldparticles (right image), contacted with 50 nm PAA-PAH coated goldparticles (left image), or control pellets of cells not contacted withthe gold nanoparticles (center image).

FIG. 5 is a drawing showing methods for producing a polyethyleneglycol-coated FB50 antibody conjugated gold nanoparticles and injectingthe nanoparticle into a subject having hepatocellular carcinoma (HCC).The FB50 antibody conjugated to the nanoparticle specifically targets anantigen of HCC and allows for imaging of a HCC tumor site using spatialharmonic imaging. The drawing shows contacting a gold nanoparticle(AuNP) with a bi-functional polyethylene glycol (PEG) having carboxylicacid functional groups (—COOH). The PEG undergoes facile attachment tothe external surface of the nanoparticle (Au-PEG). The gold nanoparticlehaving attached PEG was bio-conjugated to FB50 antibody usingcarbodiimide-amine (EDC/NHS) linking chemistry. The polyethylene glycolcoated, FB50 antibody conjugated gold nanoparticles (Au-PEG-FB50) wereinjected into a mouse having HCC, and the FB50 antibody component of theconjugated nanoparticle specifically bound to an antigen of HCC. Spatialharmonic imaging of the mouse resulted in imaging of the HCC tumor boundto the polyethylene glycol-coated FB50 antibody-conjugated goldnanoparticles.

FIG. 6 panels A-B are graphs of calculation of overall diameter ofcoated or conjugated 50 nm gold nanoparticles, and uptake of the coatedor conjugated ten nm gold nanoparticles by FOCUS cell pellets or byNIH/3T3 (a fibroblast cell line) cell pellets. FB50 antibody (specificfor an antigen of HCC) or a control antibody specific for Murutucutropical antibody (MUK) were conjugated to the nanoparticles. Data showthat the PEG-coated, FB50 antibody conjugated nanoparticles produced bymethods herein were effectively and specifically bound to andtransported into the FOCUS cell pellets and not NIH/3T3 cell pellets.

FIG. 6 panel A is a line graph of the overall average diameter in nm of:PEG-coated 50 nm gold nanoparticles (AU-PEG; middle peak), PEG-coatedFB50 antibody conjugated 50 nm gold nanoparticles (AU-PEG-FB50; rightmost peak); and control 50 nm nanoparticles that were neither coatedwith PEG nor conjugated with FB50 (AU; left most peak). Data show thatthe diameter of the FB50 antibody conjugated 50 nm gold nanoparticles(93.9±12.1 nm) was larger than PEG-coated 50 nm gold nanoparticles(81.8±12.1 nm) and control gold nanoparticles (71.3±10.0 nm).

FIG. 6 panel B is a bar graph showing average percent uptake (Average %yield) of FOCUS cell pellets (checkered; three left columns) and NIH/3T3cell pellets (solid; two right columns) contacted with conjugated ten nmgold nanoparticles. The FOCUS cell pellets were contacted with one of:PEG-coated ten nm gold nanoparticles (FOCUS+AU-PEG; left most column);PEG-coated FB50 antibody-conjugated ten nm gold nanoparticles(FOCUS+AU-PEG-FB50; second column from left); or PEG-coated MUKantibody-conjugated 50 nm gold nanoparticles (AU-PEG-FB50; third fromthe left). The NIH/3T3 cell pellets were contacted with either:PEG-conjugated ten nm gold nanoparticles (FOCUS+AU-PEG; second columnfrom the right); or PEG-coated FB50 antibody-conjugated ten nm goldnanoparticles (FOCUS+AU-PEG-FB50; right most column). Data show that atleast thirty fold more of the PEG-coated FB50 antibody conjugated ten nmgold nanoparticles were transported into the FOCUS cell pellets thaninto the NIH/3T3 cell pellets.

FIG. 7 panels A-B are a set of photographs, X-ray scatter images, andabsorption images of mice injected in vivo either with PEG-coated 50 nmgold nanoparticles or with saline (negative control). Mice were injectedin the tail vein twice in 24 hours and were sacrificed 48 hours afterthe first injection. Livers of subjects injected with the PEG-coated 50nm gold nanoparticles showed a significant average signal enhancement(23.0±14.1%) compared livers from subjects injected with saline only.

FIG. 7 panel A is a set of photographs (left column) and X-ray scatterimages (right column) of livers of subjects injected with either saline(top row) or with PEG-coated 50 nm gold nanoparticles (AU-PEG; bottomrow). The sizes of the livers for the subjects injected with saline orwith nanoparticles were comparable. Improved X-ray scatter image clarityand sensitivity were observed in livers of subjects injected withPEG-coated 50 nm gold nanoparticles (FIG. 7 panel A right column bottomrow) compared to subjects injected with saline only (FIG. 7 panel Aright column top row).

FIG. 7 panel B is a set of total absorption images (left) and a X-raytotal scatter images (right) of excised livers from subjects injectedwith either saline (top image) or PEG-coated 50 nm gold nanoparticles(AU-PEG; bottom image). The X-ray scatter images (right) more clearlydelineated the actual size of the livers for the subjects compared tothe absorption images (left), and the nanoparticles enhanced the imagingof the livers compared to the saline.

DETAILED DESCRIPTION

Nanoparticles are structures/particles that have an approximate size ofone nm to 100 nm and are used in applications, including addition tosurfaces or fluids for catalytic reactions, self-cleaning andantibacterial products, glass dyeing, sunscreen lotions andmanufacturing of optical components (e.g., optical fibers). See Raj alaet al. U.S. Pat. No. 8,231,369 issued Jul. 31, 2012. Nanoparticles arecomposed of a variety of materials including metals, metal oxides, MRIagents, semiconductors, and polymers, and possess unique characteristicsbecause of their small size.

Laboratory-scale and industrial-scale techniques are used to manufacturenanoparticles having for example a specific size distribution(mono-dispersivity), anti-agglomeration, and homogeneity (see Davis etal. U.S. Pat. No. 8,263,035 issued Sep. 11, 2012; Magdassi et al. U.S.Pat. No. 8,227,022 issued Jul. 24, 2012; and Fiannaca et al. U.S. patentapplication number US 2008/0050592 A1 published Feb. 28, 2008).Nanoparticles are produced for example by wet chemical processes and byvapor phase processes (see Murphy et al. U.S. Pat. No. 8,241,922 issuedAug. 14, 2012; and Brooks et al. U.S. Pat. No. 7,985,398 issued Jul. 26,2011). Vapor phase processes (also known as aerosol reactor processes)use a number of different devices and techniques including: flamereactors, hot-wall reactors, plasma reactors, gas condensation methods,laser ablation and spray pyrolysis (see Jun et al. U.S. Pat. No.7,988,761 issued Aug. 2, 2011; and Yang, Y. et al. 2006 CatalysisCommunications 7:281-284) to synthesize nanoparticles of controlled sizeand composition.

Nanoparticles composed of metals are used as imaging agents that areimaged using a number of different techniques including MRI,ultrasonography, and X-ray computer tomography (see Grinstaff et al.U.S. Pat. No. 5,505,932 issued Apr. 9, 1996). Imaging of nanoparticlescontaining for example gadolinium, dysoprium, manganese, iron, andplatinum involves specialized excitation and data manipulation ofdetected radiofrequency signals from non-zero spin nuclei which have anon-equilibrium nuclear spin state distribution. (Weiler et al. U.S.Pat. No. 6,370,415 issued Apr. 9, 2002; and Weiler et al. U.S. Pat. No.6,595,211 issued Jul. 22, 2003). In conventional MRI the nucleiresponsible for the detected signals are protons (e.g., protons ofwater), and the non-equilibrium spin state distribution is achieved byplacing the subject in a strong magnetic field (to enhance thepopulation difference between the proton spin states at equilibrium),and then exposing the subject to pulses of radiofrequency radiation atthe proton Larmor frequency, which excites spin state transitions andcreates a non-equilibrium spin state distribution (see Seri et al. U.S.Pat. No. 5,811,077 issued Sep. 22, 1998).

Nanoparticles such as gold nanoparticles are potential contrast agentsfor X-ray imaging because the nanoparticles are constructed to benon-toxic and to have a higher atomic number and X-ray absorptioncoefficient than typical iodine-based contrast agents (Hainfeld, J. F.et al. 2006 Br. J. Radiol. 79: 248-253; Kim, D. et al. 2007 J. Am. Chem.Soc. 129: 7661-7665; and Kojima, C. et al. 2010 Nanotechnology 21:245104). Biodistribution for example of small gold nanoparticlesinjected intravenously is detected by X-ray imaging (Hainfeld, J. F. etal. 2006 Br. J. Radiol. 79: 248-253). Gold nanoparticles approximately30 nm in diameter have been injected intravenously and used for in vivocomputed tomography (CT) imaging of hepatoma in the liver (Kim, D. etal. 2007 J. Am. Chem. Soc. 129: 7661-7665). However, successful CT scanand MRI imaging of tissues required in vivo injection of largequantities of gold nanoparticles, or creating a high density of suchparticles at the image target for example, by linking the nanoparticlesto targeted delivery vehicles.

Without being limited by any particular theory or mechanism of action,it is here envisioned that the spatial frequency heterodyneimaging/spatial harmonic imaging techniques described in Examples hereinhave the advantage of using a much reduced amount of nanoparticles(e.g., gold nanoparticles) to produce a visibly-enhanced contrastcompared to typical absorption based X-ray imaging. Additionally, theimaging described herein using nanoparticles provides a nearlybackground-free image because scattered x-radiation is very wellseparated from transmitted radiation due to the different angles atwhich the scattered radiation reaches a detector.

X-rays employed for medical diagnostic imaging are electromagneticradiation of approximately 0.01 nm to ten nm wavelength and high energy,approximately 100 electronvolts to 100 kiloelectronvolts (keV) andtypically about two keV to 50 keV. A beam of X-rays directed to an atomis absorbed or deflected. The deflected X-rays define extent of scatter.Compton scattering describes an incident X-ray photon that is deflectedfrom its original path by an electron. Scatter traditionally serveslittle purpose in imaging of tissues and patients, as scattering yieldsa diffuse signal that reduces contrast and clarity of the image(Feldmesser et al. U.S. Pat. No. 6,529,582 issued Mar. 4, 2003). Tissueimages are generally prepared using only X-rays passed directly throughthe patient without colliding with atoms along the path. At a givenpoint of the image plane or detector, the quantity of X-rays at thatpoint indicates the degree of absorption of the primary beam in thepatient on the line from the X-ray source to the X-ray receptor (e.g.,the film). The scattered X-rays arrive at the X-ray film from variousangles and places in the body not related to the path from the source tothe receptor. Thus unwanted scattered X-rays cause the image to bedistorted and cloudy. These distortions in the image from scatteringresult in reduced image contrast, and obscure the small variations inX-ray absorption that exist within the body of a subject, for whichimages should be obtained for an accurate diagnosis or prognosis.

Spatial frequency heterodyne imaging (also known as spatial hainionicimaging) was used in examples herein with an X-ray scatter reducing gridfor absorbing rays scattered when radiation was transmitted through thesubject, and to obtain a high quality image to reduce or eliminatescatter radiation. The grid was placed in the direction of propagationof an X-ray beam for example in parallel and in the shape of a flatplate or box. Radiation was transmitted through the grid and to a sampleor a subject, such that the scattered radiation traveled obliquely andwas absorbed and reduced by the radiation-absorbing portions. Theprimary radiation was transmitted (substantially linearly) through avial containing the sample or through the subject. The primary radiationtransmitted through the radiation-transmitting portions (e.g., wood, ametal, a plastic, and voids) of the grid and then the sample reached adetector that formed a radiation-transmitted image. Theradiation-absorbing portions in certain embodiments are formed from anabsorbing material or a dense shield material that attenuatesx-radiation such as lead or the like. The radiation-transmittingportions and radiation-absorbing portions were in certain embodimentsalternately or closely arranged, e.g., symmetrical positions. Theradiation-transmitting portions have a high transmittance to avoidreducing transmission of the primary radiation to the sample and thedetector (see Ogawa U.S. Pat. No. 6,707,884 issued Mar. 16, 2004; Stein,A. F. et al. 2010 Opt. Express 18: 13271-13278; Wen, H. et al. 2009Radiology 251: 910-918; and Wen, H. et al. 2008 IEEE Trans Med Imaging27: 997-1002, each of which is incorporated by reference herein in itsentirety). Fourier transformation was is used to process the imageproduced by the detector (FIG. 1).

An imaging technique described herein involves in certain embodimentsuse of surface-modified gold nanoparticles and spatial frequencyheterodyne imaging. The nanoparticles (containing at least one of ametal or a metal oxide) are modified in certain examples by attaching abinding agent and/or a polymer (e.g., a polyelectrolyte) layer. Thenanoparticles were observed to be biocompatible and non-toxic and weretaken up by cells.

Nanoparticles used in various embodiments of the invention wereconstructed using layer-by-layer coatings of polymers such aselectrolytes. Layer-by-layer coating of polyelectrolytes is a versatilemethod for modifying the surface chemistry of nanoscale materialsincluding gold nanoparticles and nanorods (Mayya, K. S. et al. 2003 Adv.Funct. Mater. 13: 183-188; Gittins, D. I. et al. 2001 J. Phys. Chem. B.105: 6846-6852; Gole, A. et al. 2005 Chem. Mater. 17: 1325-1330; andMurphy, C. J. 2005 Chem. Mater. 17: 1325-1330). Layer-by-layer coatingsof charged polyelectrolytes are useful to stabilize colloidalsuspensions.

Deposition of polyelectrolyte coatings (e.g., poly(acrylic acid) andpoly(allylamine hydrochloride) on surfaces of structures under specificpH conditions result in the ability of the coated surfaces to bind cells(Mendelsohn, J. D. et al. 2003 Biomacromolecules 4: 96-106).Poly(acrylic acid) and poly(allylamine hydrochloride) are weakpolyelectrolytes with different degrees of ionization, and as a resulteach of these electrolytes has a strength of electrostatic interactionthat is pH-dependent (Shiratori, S. S. et al. 2000 Macromolecules 33:4213-4219). Poly(acrylic acid) stock solutions and poly(allylaminehydrochloride) stock solutions used in certain embodiments herein wereadjusted to have a pH of 8.4 and 3.7, respectively, and under theseconditions poly(acrylic acid) has a pKa of about 4.5 and poly(allylaminehydrochloride) has a pKa about 8.5. Stock solutions of poly(acrylicacid) and poly(allylamine hydrochloride) described herein were fullycharged, i.e., anionically and cationically, respectively.

The de-protonated carboxylic acid groups of anionic poly(acrylic acid)(PAA) interacted strongly with protonated amine groups of cationicpoly(allylamine hydrochloride) (PAH), such that the oppositely chargedlayers produced thin, flat coatings on the nanoparticle surface. Wateris unable to penetrate the surface layer of the PAA-PAH coatednanoparticle because the PAA-PAH polyelectrolyte layers were so tightlycross-linked by the anionic and cationic (i.e., ionic) interactions. Theelectrolyte coated nanoparticles produced by this method thereforeresulted in a very hydrophobic polymer barrier to a surrounding aqueousenvironment. The hydrophobicity of the electrolyte coating resulted innanoparticle surfaces that were cytophilic (Lee, J. H. et al. 1995 Prog.Polym. Sci. 20: 1043-1079; Alexis, F. et al. 1998 Mol. Pharm. 5:505-515; Brandenberger, C. et al. 2010 Small 6: 1669-1678). Furthermore,the thickness and specificity of the polyelectrolyte coating iscontrolled by varying and optimizing the number of layers deposited onthe surface of the nanoparticles (Mayya, K. S. et al. 2003 Adv. Funct.Mater. 13: 183-188; and Gittins, D. I. et al. 2001 J. Phys. Chem. B.105: 6846-6852). Without being limited by any particular theory ormechanism of action, it is here envisioned that thepolyelectrolyte-coated nanoparticles described herein were effectivevehicles for cellular uptake by mechanisms such as phagocytosis andendocytosis.

Specificity of the nanoparticles was further enhanced by conjugating avariety of antibody binding agents to the polyelectrolyte coatings.Layer-by-layer coating and antibody binding agent conjugation ofnanoparticle composition functionalized the nanoparticle compositionsand increased the specific binding of the nanoparticles to differenttypes of cells and tissues (Murphy, C. J. 2005 Chem. Mater. 17:1325-1330). Phagocytosis of the polymer coated gold nanoparticles inliving cells was measured in a model system using FOCUS cells, a humanhepatocellular carcinoma (HCC) cell line (He, L. et al. 1984 In Vitro20: 493-504). FOCUS cells are a model for the study of targeted imagingof cells because these cells express specific HCC antigens that arerecognized and bound by binding agents such as monoclonal antibodies(Hurwitz, E. et al. 1990 Bioconjugate Chem. 1: 285-290; Takahashi, H. etal. 1989 Gastroenterology 96: 1317-1329; Mohr, L. et al. 2004Gastroenterology 127: S225-S231; and Luu, M. et al. 2009 Hum. Pathol.40: 639-644). A strong antibody-antigen interaction exists between FB50antibody and aspartyl (asparaginyl)-β-hydroxylase, a proteinover-expressed in liver cancer cells such as HCC (Luu, M. et al. 2009Hum. Pathol. 40: 639-644).

Examples herein used antibody-conjugated nanoparticles and spatialfrequency heterodyne imaging to directly target and image tumors in ananimal model (Mohr, L. et al. 2004 Gastroenterology 127: S225-S231; andLuu, M. et al. 2009 Hum. Pathol. 40: 639-644). The N-terminal (aminoterminus) of FB50 antibody was conjugated to the outer surface of a PEGcoated nanoparticle by 1-ethyl-3-3-dimethylaminopropyl]carbodiimide andamine (EDC/NHS) cross-linking chemistry (FIG. 5). In certainembodiments, the polymer (e.g., PEG, PAA, and PAH) coatings are mixedwith and bound to the antibody binding agent prior to being contactedwith the nanoparticle to produced a polymer-coated antibody-conjugatednanoparticle, which can be used for example to binding normal tissues orcancer tissues in vivo (FIGS. 6-7). In certain embodiments, the bindingagent (e.g., protein) or plurality of binding agents is conjugated orattached to the nanoparticles at a carboxy-terminus or anamino-terminus.

Nanoparticles are coated with a polyethylene glycol (PEG) to preventnonspecific protein adsorption, to reduce nonspecific cellular uptake,and to increase the circulation times of nanoparticles in thebloodstream (Alexis, F. et al. 1998 Mol. Pharm. 5: 505-515;Brandenberger, C. 2010 Small 6: 1669-1678; Hucknall, A. et al. 2009 Adv.Maert. 21: 2441-2446; and Lipka, J. et al. 2010 Biomaterials 31:6574-6581). In certain embodiments, the PEG polymer layer was coated togold nanoparticles using layer-by-layer coating techniques and then usedto conjugate binding agents to the nanoparticlesthat target specificcancers in vivo (FIG. 5). The gold nanoparticles were then used todeliver gold to the HCC tumors, and the cells and the nanoparticles wereimaged and diagnosed.

Cancer cells such as FOCUS cells were targeted in vivo by contacting thecells with nanoparticles conjugated with an FB50 antibody binding agentthat binds to HCC antigens. The binding agent-conjugated nanoparticlescontact the cells and/or or deliver therapeutic agents to the targetedcells, and avoid being taken up by healthy cells. Spatial frequencyheterodyne imaging was performed on samples of pellets of FOCUS cellsincubated with different amount of PAA-PAH coated gold nanoparticles(ten nm or 50 nm diameters). Control samples were incubated in absenceof gold nanoparticles. Each set of pellets was imaged multiple times toexclude false signals due to possible non-uniformities of thesensitivity of the imaging system. A vial holder was designed and usedto keep vials containing cell samples and nanoparticles in a positionthat could be reproduced. Apertures of the same size were drilledthrough a thin block of aluminum so that the vials, placed into theseapertures, were positioned at exactly the same position and height.

Examples herein in certain embodiments used cell pellets each containingapproximately 10⁷ FOCUS cells. The pellet size of FOCUS cellscorresponded to the approximate size of a small tumor (severalmillimeters in diameter). The FOCUS cells were cultured and incubatedwith gold nanoparticles (ten nm and 50 nm diameter). Beams of X-rayswere directed through an absorption grid and a vial containing thesample of cells, and the scatter X-ray radiation was detected using acharge-coupled camera (CCD). Fourier transformation of the X-ray scatterdata produced by the spatial frequency heterodyne imaging yielded clearand sensitive images of the cells. Data herein show that spatialfrequency heterodyne imaging of cells and tissues contacted with thenanoparticles was more sensitive than typical absorption-based imagingtechniques, for example, tissues imaged with CT scans and MRI imaging.

Data show enhanced FOCUS cell phagocytosis of the gold nanoparticleshaving a bi-layer of PAA and the cationic PAH compared to phagocytosisof gold nanoparticles having no coating (FIG. 4). Presence of thepolymer layers of polyelectrolytes on the nanoparticle surface resultedin nearly double amount of uptake of the nanoparticles by the FOCUScells (see Table 1). The mass of gold taken up by each cell correspondsto several hundred 50 nm gold nanoparticles and tens of thousands of tennm gold nanoparticles (Table 2).

TABLE 1 Cellular uptake of gold nanoparticles. uncoated 10 nm 50 nm 10nm PAA-PAH PAA-PAH nanoparticles nanoparticles nanoparticles mass ofgold taken up per cell 1.2 ± 0.5 2.8 ± 0.4 2.2 ± 0.3 (picograms)approximate number of 108,000 275,000 1730 nanoparticles per cellapproximate volume fraction 0.00025% 0.00063% 0.00049% of nanoparticlesin each cell

TABLE 2 Average signal enhancements of cells due to gold labelingincubation with PAA-PAH gold nanoparticles. replicate 1 replicate 2 massof gold taken up per cell (pg) 0.45 ± 0.09 0.76 ± 0.11 average number of10 nm 44,600 75,200 nanoparticles per cell 50 nm 356 602 change insignal per original image 1.3 ± 4.4 1.1 ± 3.0 pellet (%) ^(a) processedimage 1.6 ± 0.3 4.4 ± 0.8 signal enhancement original image 2.9 ± 9.81.4 ± 3.9 per 1 pg of gold taken processed image 3.6 ± 0.7 5.7 ± 1.1 upper cell (%) approximate potential signal enhancement 11 ± 2  17 ± 3 for a pellet of 10⁷ cells (%) ^(a) All enhancements are reported inlogarithm scale.

A set of representative X-ray scatter imaging photographs of the pelletsunder cell culture medium shows gold labeling by the nanoparticles (seeFIG. 4). Processed X-ray scatter images using spatial frequencyheterodyne imaging showed significant image enhancement due to goldlabeling by the polymer modified nanoparticles that was almost five-foldgreater than enhancements identified in the absorption images (Table 2).Further, examples herein using polymer coated antibody-conjugated goldnanoparticle compositions produced data showing increased cellularuptake of gold nanoparticles into cells and tissues compared to resultsfor cells contacted with only polymer coated gold nanoparticles (FIG.6). For example, data herein showed that less than 0.001% of each cellvolume is occupied by polymer coated gold nanoparticles, allowing forsignificantly increased amounts of gold nanoparticles to be furtherintroduced into each cell, thereby increasing scatter signal and,ultimately, enhancing visibility in scatter images. The sensitivity andpotential specificity of the nanoparticle-based imaging techniquesdescribed herein show that these are effective methods, compositions andkits for early detection and diagnosis of cancers such as HCC.

Examples herein show that cell pellet samples of FOCUS cellsphagocytosed gold nanoparticles of different sizes, and that the goldnanoparticle-containing cell pellets of FOCUS cells were distinguishablefrom cell pellets of FOCUS devoid of gold nanoparticles using spatialfrequency heterodyne imaging. Spatial frequency heterodyne images ofFOCUS cell pellets labeled with gold nanoparticles showed that greaterthan 85% of the images indicate a signal enhancement compared to X-rayscatter images of FOCUS pellets containing no gold. Data herein showedthat each FOCUS cell phagocytosed approximately three picograms of goldnanoparticles per cell (Table 1), and that X-ray scatter imagingenhances visibility by up to 5.7% for every picogram of gold in thecells (Table 2). Without being limited by any particular theory ormechanism of action, it is here envisioned that that spatial frequencyheterodyne imaging enhanced visibility of gold nanoparticle-labeledcells by more than 17% on a logarithmic scale.

Examples herein obtained images of cell pellets that were severalmillimeters in diameter, which corresponds to the approximate size of asmall tumor. Spatial frequency heterodyne imaging of nanoparticles wasused in Examples to detect tumor-sized cell samples that aresignificantly below the detection limits of current imaging techniquesfor HCC and many other cancers. Currently cancers such as HCC goundiagnosed until the tumors are several centimeters in size, viz., thecancers are detected only upon reaching a size an order of magnitudegreater than the sizes detected using the methods described herein forX-ray scatter imaging of nanoparticles. Thus, methods, compositions, andkits described herein were effective for imaging and for differentiatingcancerous tissues and normal tissues.

Spatial frequency heterodyne imaging was performed on cell pelletsamples that were placed under water in an in vivo model, because waterand tissues (e.g., liver tissues) share a similar radiological density.Data showed that in vitro X-ray scatter imaging of the submerged cellsand nanoparticles was effective in visualizing the cells. Without beinglimited by any particular theory or mechanism of action, it is hereenvisioned that spatial frequency heterodyne imaging of nanoparticlescontaining a metal as described herein would detect small in situ tumors(less than a few millimeters in size) in liver and in other internalorgans and tissues.

Metal nanomaterials (containing for example gold) have been used forcancer therapy applications (e.g., introduction of bioactive agents) aswell as imaging applications (see Sung et al. U.S. Pat. No. 7,985,426issued Jul. 26, 2011). For example, gold nanoshells have been developedfor use in photothermal cancer therapy (Hirsch, L. R. et al. 2003 Proc.Natl. Acad. Sci. U.S.A. 100: 13549-13544; Choi, M. et al. 2007 S. NanoLett. 7: 3759-3765; Lal, S.; Clare, S. E. et al. 2008 Ace. Chem. Res.41: 1842-1851). These nanoshells composed of metals and metal oxideshave highly tunable plasmon resonances, allowing strong absorption oflight even under the circumstances of the frequency of the incominglight matching plasmon oscillation frequencies of the nanoshells. Thenanoshells convert energy absorbed from directed light into heat,killing cells containing the nanoshells and leaving unharmed thesurrounding unlabeled cells and tissues. Clinical trials of therapiesusing nanoshells and nanostructures are being investigated (Clare, S. E.et al. 2008 Acc. Chem. Res. 41: 1842-1851).

Gold nanoparticles have also been used to provide dose enhancement incancer radioablation therapy. Small gold nanoparticles have for examplebeen injected intravenously such that the nanoparticles accumulated intumors and improved X-ray therapy at the tumor site (Hainfeld, J. F.2004 Physics in Medicine and Biology 49: N309-N315). As both of thetherapeutic applications discussed above use electron-densenanomaterials, it is envisioned herein that the X-ray scatter imagingmethods and compositions described herein would be effective incombination with these and similar applications for the dual imaging andtherapy of cells and tissues.

An aspect of the invention provides a composition for enhanced imagingand/or diagnosing a cells or a tissue for example a tumor, thecomposition including a nanoparticle having at least one polymer layercoating the nanoparticle, such that the nanoparticle binds to and/or isphagocytosed by the cells or the tissue to enhance visibility of thetumor by X-ray scatter imaging.

In an embodiment of the composition, the nanoparticle includes a metal,for example the metal is at least one of: silver, copper, gold, mercury,cadmium, zinc, nickel, palladium, platinum, rhodium, mercury. In anembodiment of the composition, the metal is a transition metal forexample titanium or iron.

In an embodiment of the composition, the nanoparticle includes a shellof a material for example a metal or a carbon, surrounding a core of amaterial with an electron density lower than that of the shell. Forexample, a carbon core or a silica core is surrounded by a layer of goldor another suitable metal, metal oxide, MRI agent, inorganic material,or organic material.

In an embodiment of the composition, the nanoparticle is at least aboutfive nm in diameter. In various embodiments of the composition, thenanoparticle has a diameter of at least about: two nm, five nm, ten nm,20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, and 100 nm. Inan embodiment, the nanoparticle includes a plurality of nanoparticleshaving an average diameter greater than about five nm.

In an embodiment of the composition, the nanoparticle is less than about100 nm in diameter. In various embodiments of the composition, thenanoparticle includes a diameter less than about: 100 nm, 90 nm, 80 nm,75 nm, 65 nm, 60 nm, 50 nm, 45 nm, 40 nm, 30 nm, 25 nm, 20 nm, 15 nm,and ten nm. In an embodiment, the nanoparticle includes a plurality ofnanoparticles having an average diameter less than about 100 nm.

In an embodiment of the composition, the polymer layer includes at leastone of: an anionic polyelectrolyte, a cationic polyelectrolyte, and apolyethylene glycol. In an embodiment of the composition, thepolyethylene glycol polymer is about 10,000 average molecular weight insize. In other embodiments, the polyethylene glycol polymer includes anaverage molecule weight in size of at least one of: about 1,000; about2,000; about 4,000; about 6,000; about 8,000; about 12,000; about15,000; about 20,000; and about 30,000.

In an embodiment of the composition, the polymer layer includes ananionic polyelectrolyte. In various embodiments of the composition, theanionic polyelectrolyte is at least one selected from: a poly(acrylicacid), a 4-Styrenesulfonic acid,poly(2-acrylamido-2-methyl-1-propanesulfonic acid), apoly(2-acrylamido-2-methyl-1-propanesulfonic acid, apoly(4-styrenesulfonic acid), a poly(4-styrenesulfonic acid-co-maleicacid), a polyanetholesulfonic acid, a poly(vinylsulfonic acid), and asalt thereof. In various embodiments of the composition, the polymerlayer is a bio-compatible layer or a non-toxic-layer.

In an embodiment of the composition, the polymer layer includes acationic polyelectrolyte. In various embodiments of the composition, thecationic polyelectrolyte is at least one selected from: adiallyldimethylammonium, a poly(acrylamide-co-diallyldimethylammoniumchloride), a poly(allylamine hydrochloride, apoly(diallyldimethylammonium chloride), and a salt thereof.

In an embodiment of the composition, the anionic polyelectrolyte and/orthe cationic polyelectrolyte include an anionic poly(acrylic acid) andan cationic poly(allylamine hydrochloride), respectively.

The composition further includes for example at least one binding agentthat selectively binds the nanoparticle to the tissue, for example, thebinding agent includes at least one selected from the group of: a drug,a protein such as an antibody or a binding protein, a carbohydrate suchas a sugar, and a nucleotide sequence. For example, the antibody is apolyclonal antibody, a monoclonal antibody, or a portion thereof forexample a Fv; a Fab; a Fab′; or a F(ab′)₂. In an embodiment of thecomposition, the antibody includes a fusion protein or a chimericprotein.

The tumor in various embodiments of the composition is located orassociated with a site or cancer selected from the group of: melanoma;sarcoma; carcinoma (e.g., colon and hepatocellular); pancreatic;lymphoma; glioma; lung; esophagus; mammary; prostate; head; neck;ovarian; kidney; and liver.

An aspect of the invention provides a composition including: a goldnanoparticle; a polymer layer coating the nanoparticle comprising forexample a polyethylene glycol or a polyelectrolyte such as an anionicpoly(acrylic acid), and a cationic poly(allylamine hydrochloride); and,a monoclonal antibody or portion thereof that specifically binds a tumorantigen and is bound to the polymer layer.

In an embodiment of the composition, the nanoparticle is at least aboutfive nm in diameter. In an embodiment of the composition, thenanoparticle is less than about 100 nm in diameter. For example, thenanoparticle is about ten nm or about 50 nm in diameter.

In an embodiment of the composition, the monoclonal antibody is FB50 andthe tumor antigen is aspartyl (asparaginyl)-β-hydroxylase. In anembodiment of the composition, the tumor antigen is associated withhepatocellular carcinoma.

An aspect of the invention provides a method of diagnosing a presence ofa tumor in a subject including: contacting a tissue with a compositionhaving: a gold nanoparticle; a polymer layer coating the nanoparticle,such that the polymer layer comprises for example a polyethylene glycolor a polyelectrolyte such as an anionic poly(acrylic acid) or a cationicpoly(allylamine hydrochloride); and, a binding agent that specificallybinds a tumor antigen, such that the binding agent is bound to thepolymer layer; and, detecting the presence or absence of the tumorattached to the nanoparticle using an imaging device, such thatdetecting comprises identifying presence or accumulation thenanoparticles in the tissue by X-ray scatter imaging to detect thetumor. In an embodiment of the method, the tissue is in situ or in vivo.Alternatively, the tissue is in vitro, for example the method in anembodiment includes, prior to contacting the tissue with thecomposition, collecting or obtaining the tissue from the subject.

In various embodiments of the method, the imaging device includes aX-ray device or a MRI device. In various embodiments of the method,detecting using the X-device involves generating X-rays using an X-raytube or laser or accelerator generated X-rays.

In an embodiment of the method, detecting the presence of the tumorincludes spatial frequency heterodyne imaging or spatial harmonicimaging, for example the spatial harmonic imaging involves irradiatingwith an X-ray source and detecting with an absorption grid, and/or adetector. In an embodiment of the method, detecting the presence of thetumor involves identifying in situ the presence of the tumor.Alternatively, detecting involves identifying in vitro the presence ofthe tumor.

In various embodiments of the method, the nanoparticle includes at leastone of: silver, copper, gold, mercury, cadmium, zinc, nickel, palladium,platinum, rhodium, mercury, and a combination thereof.

In various embodiments of the method, the anionic poly(acrylic acid) isdirectly contacting the nanoparticle, and the cationic poly(allylaminehydrochloride) and the polyethylene glycol coat the anionic poly(acrylicacid).

The method in an embodiment further includes prior to contacting thetissue, preparing the nanoparticle by coating the nanoparticle with thepolymer layer and attaching the binding agent.

In various embodiments of the method, the tissue includes a plurality ofcells selected from the group of: epithelial cells, hematopoietic cells,stem cells, spleen cells, kidney cells, pancreas cells, liver cells,neuron cells, glial cells, smooth or striated muscle cells, sperm cells,heart cells, lung cells, ocular cells, bone marrow cells, fetal cells,peripheral blood mononuclear cells, leukocyte cells, lymphocyte cells,and living postmitotic cells.

In an embodiment of the method, the binding agent includes at least oneselected from the group of: a drug, a protein, a carbohydrate, and anucleotide sequence. In an embodiment of the method, the binding agentincludes a polyclonal antibody or a portion thereof. In an embodiment ofthe method, the binding agent includes a monoclonal antibody or aportion thereof. In an embodiment of the method, the antibody isspecific for the tumor antigen selected from the group of: aspartyl(asparaginyl)-β-hydroxylase, alphafetoprotein, carcinoembryonic antigen(CA), CA-125, mucin 1, epithelial tumor antigen, tyrosinase,melanoma-associated antigen, tumor protein 53, human chorionicgonadotropin, vimentin, CD34, desmin, and glial fibrillary acidicprotein. For example, the binding agent is monoclonal antibody FB50 orSF25. In various embodiments of the method, the antibody is animmunoglobulin selected from the group consisting of: IgA, IgD, IgE, andIgG. In an embodiment of the method, the antibody is from at least oneorigin selected from: human, murine, ovine, bovine, feline, canine,hircine, and equine.

In various embodiments of the method, the tumor is selected from thegroup of: melanoma; colon carcinoma; pancreatic; lymphoma; glioma; lung;esophagus; mammary; prostate; head and neck; ovarian; kidney; liver, andhepatocellular carcinoma.

The method in an embodiment further includes therapeutically treatingthe tumor, for example to surgically excising the tumor or to reducesize of the tumor. In various embodiments, the method further includesadministering a therapeutic agent to the tumor or to the subject. Invarious embodiments, the therapeutic agent is at least one of: ananti-cancer, anti-tumor, an anti-proliferative, and ananti-inflammatory.

An aspect of the invention provides a method of manufacturing acomposition for imaging and/or diagnosing a tumor involving: coating ananoparticle with a polymer layer, such that the polymer layer coats thenanoparticle and includes for example a polyethylene glycol and/or apolyelectrolyte such as an anionic poly(acrylic acid) or a cationicpoly(allylamine hydrochloride), to obtain a resulting polymer-coatednanoparticle; and contacting the resulting polymer-coated nanoparticlewith at least one binding agent that selectively attaches to the polymerlayer and binds the tumor, thereby producing the composition for imagingand/or diagnosing the tumor. In an embodiment, the polymer layerincludes both the polyethylene glycol and the polyelectrolyte layer.

In an embodiment of the method, the binding agent that binds the tumorincludes at least one selected from the group of: a drug, a protein, acarbohydrate, and a nucleotide sequence.

In an embodiment of the method, the binding agent includes a monoclonalantibody that specifically targets the tumor or a surface protein orpeptide located on the tumor. For example, the binding agent ismonoclonal antibody FB50 or monoclonal antibody SF25.

In an embodiment of the method, the antibody includes a polyclonalantibody that specifically targets the tumor or a surface protein orpeptide located on the tumor.

In various embodiments of the method, the binding agent binds a tumorantigen that is at least one selected from the group of: aspartyl(asparaginyl)-β-hydroxylase, alphafetoprotein, carcinoembryonic antigen(CA), CA-125, mucin 1, epithelial tumor antigen, tyrosinase,melanoma-associated antigen, tumor protein 53, vimentin, CD34, desmin,and glial fibrillary acidic protein.

In various embodiments of the method, the tumor is selected from thegroup of melanoma; colon carcinoma; pancreatic; lymphoma; leukemia;glioma; lung; esophagus; mammary; prostate; head and neck; ovarian;kidney; liver, and hepatocellular carcinoma.

The method in an embodiment includes prior to contacting thenanoparticle with at least one polymer layer, constructing thenanoparticle to have a diameter greater than about five nm in diameter,or to have a diameter less than about 100 nm in diameter.

An aspect of the invention provides a kit for diagnosing presence of atumor in a subject with a composition, the kit including: ananoparticle; a polymer layer coating the nanoparticle, having: apolyethylene glycol or a polyelectrolyte such as an anionic poly(acrylicacid) and/or a cationic poly(allylamine hydrochloride); and, a bindingagent that specifically binds a tumor antigen, wherein the antibody isbound to the polymer layer; the kit further including instructions foruse and a container.

In an embodiment of the kit, the nanoparticle comprises a metal, forexample a transition metal. In various embodiments of the kit, the metalincludes at least one of: silver, copper, gold, mercury, cadmium, zinc,nickel, palladium, platinum, rhodium, mercury, and a combinationthereof.

In an embodiment of the kit, the anionic poly(acrylic acid) contacts thenanoparticle, and the cationic poly(allylamine hydrochloride) contactsthe anionic poly acrylic acid.

In an embodiment of the kit, the binding agent includes at least oneselected from the group of: a drug, a protein, a carbohydrate, and anucleotide sequence. In an embodiment of the kit, the binding agent thatselectively attaches the composition to the tumor is an antibody or aportion thereof, for example the antibody includes a monoclonal antibodyor a portion thereof. In an embodiment of the invention, the antibodyincludes a polyclonal antibody or a portion thereof. In an embodiment ofthe kit, the binding agent is monoclonal antibody FB50 or monoclonalantibody SF25, or a portion thereof.

In an embodiment of the kit, the tumor is selected from the group of:melanoma; colon carcinoma; pancreatic; lymphoma; glioma; lung;esophagus; mammary; prostate; head and neck; ovarian; kidney; liver, andhepatocellular carcinoma. In certain embodiments, the kit includes anypharmaceutical composition described herein.

In various embodiment of the kit, the tumor antigen is at least oneselected from the group of: aspartyl (asparaginyl)-β-hydroxylase,alphafetoprotein, carcinoembryonic antigen (CA), CA-125, mucin 1,epithelial tumor antigen, tyrosinase, melanoma-associated antigen, tumorprotein 53, vimentin, CD34, desmin, and glial fibrillary acidic protein.

An aspect of the invention herein provides a method of identifying in amodel system a potential therapeutic agent for treating or preventing atumor, the method including: contacting a first sample of cells ortissue having a tumor with a composition including: a gold nanoparticle;a polymer layer coating the nanoparticle comprising for example apolyethylene glycol or a polyelectrolyte, for example thepolyelectrolyte includes an anionic poly(acrylic acid) or a cationicpoly(allylamine hydrochloride); and, a monoclonal antibody thatspecifically binds a tumor and is bound to the polymer layer, contactinga second sample of the cells or tissue having the tumor with thecomposition and a potential therapeutic agent; and measuring in thefirst sample and the second sample, an amount of the marker, such thatthe marker is characteristic of the tumor, such that the amount of themarker in the second sample compared to that in the first sample is ameasure of treatment and protection by the potential therapeutic agent,such that a decreased amount of the marker in the second sample comparedto the first sample is an indication that the potential therapeuticagent is therapeutic, thereby identifying the potential therapeuticagent for treating or preventing the tumor. In an embodiment of themethod, measuring further comprises detecting the presence of the tumorin first sample and the second sample using X-ray scatter imaging, forexample spatial harmonic imaging.

An aspect of the invention provides a composition for imaging and/ordiagnosing a tumor including: nanoparticles having at least one bindingagent that binds to and/or is suitable for phagocytosis by the tumor,such that the nanoparticles are viewed by X-ray scattering imaging, forexample spatial harmonic imaging, to detect, image, or diagnose thetumor.

In an embodiment of the composition, the nanoparticles include a metalfor example at least one of: silver, copper, gold, mercury, cadmium,zinc, nickel, palladium, platinum, rhodium, mercury, or a combinationthereof, and the nanoparticles are determined to be at least about fivenm in diameter and less than about 100 nm in diameter.

In an embodiment of the composition, the nanoparticles include a coresurrounded by a metal shell with a higher electron density than thecore. For example, the core includes carbon or silica.

In an embodiment of the composition, the binding agent is specificallyattached to a portion of a surface of the nanoparticles, for example thebinding agent is attached using a linker such as an amino acid, apolymer, or a nucleotide, and such that the nanoparticles effectivelybind to the tumor and scatters X-rays once irradiated.

In an embodiment of the composition, the nanoparticles include afullerene or C₆₀ Bucky ball, and the binding agent is linked for exampleto the Bucky ball. In various embodiments of the composition, thebinding agent includes at least one selected from the group of: a drug,a protein such as an antibody or a binding protein, a carbohydrate suchas a sugar, and a nucleotide sequence. For example, the binding agentincludes a monoclonal antibody, a polyclonal, or a portion thereof.

An aspect of the invention provides a method of diagnosing a presence ofa tumor in a subject comprising: contacting a tissue with a compositioncomprising a nanoparticle having at least one binding agent that bindsto and/or is suitable for phagocytosis by the tumor; and, detecting thepresence of the tumor attached to the nanoparticle using X-ray scatterimaging for example spatial harmonic imaging. In various embodiments ofthe method, the composition further comprises any of compositionsdescribed herein.

In an embodiment of the method, detecting the presence of the tumorincludes applying a mathematical operation such as a Fourier transformto detect X-ray scatter measurements or data. In an embodiment of themethod, detecting the presence of the tumor is performed using anabsorption grid. In an embodiment of the method, the detector includes acamera such as a charge coupled device that detects the presence of thecomposition and/or nanoparticle.

An aspect of the invention provides a kit for diagnosing a presence of atumor in a subject comprising: a composition including a nanoparticlehaving at least one binding agent that binds to and/or is suitable forphagocytosis by the tumor; instructions for use; and a container. Invarious embodiments of the kit, the composition comprises any of thecompositions described herein for detecting or diagnosing the tumor byX-ray scattering imaging including for example at least one of:nanoparticles having at least one binding agent that binds to and/or issuitable for phagocytosis by the tumor; a composition having: a goldnanoparticle, a polymer layer coating the nanoparticle, such that thepolymer layer includes for example a polyethylene glycol or apolyelectrolyte such as an anionic poly(acrylic acid) or a cationicpoly(allylamine hydrochloride); and, a binding agent that specificallybinds a tumor antigen, such that the binding agent is bound to thepolymer layer. The kit in an embodiment further includes a receptacle.

The compositions, methods, kits and devices herein show an imagingtechnique for visualizing cells and/or for the early diagnosis ofcancers and tumors (e.g., hepatocellular carcinoma) usingsurface-modified nanoparticles (having an attached binding agent) andX-ray imaging. The binding agent selectively binds to for example atumor or cancer cell, and in certain embodiments is an antibody such asa monoclonal antibody, a polyclonal antibody, or a portion thereof. Incertain embodiments the monoclonal antibody is a FB50 antibody (Rand etal. 2011 Nano Lett. 11:2678-2683), or a SF25 antibody. See alsoTakahashi et al., 1988 Cancer Res 48: 6573 and Takahashi et al. U.S.Pat. No. 5,212,085 issued May 18, 1993. Cancerous issues labeled withthese electron-dense particles and imaged using spatial harmonic imagingshow enhanced X-ray scattering over normal tissues, allowing foreffectively differentiation of the cancerous cells containing thenanoparticles from normal non-cancerous cells not containing thenanoparticles. Data show both in vitro and in vivo detection of tumorsas small as a few millimeters in size.

Binding Agents

The methods of the present invention use nanoparticles and spatialfrequency heterodyne imaging in certain embodiments to image cells or atissue, and to diagnose or prognose presence or progression of a type ofcell or tissue (e.g., cancerous). The nanoparticles in embodimentsherein include a binding agent such as least one selected from the groupof: a drug, a protein, a carbohydrate, and a nucleotide sequence

In certain embodiments, the binding agent is an antibody that isattached or conjugated to the nanoparticle and selectively binds thecells or the tissue, such as a tumor. The term “antibody” as used hereinincludes whole antibodies and any antigen binding fragment (i.e.,“antigen-binding portion”) or single chains of these. A naturallyoccurring “whole” antibody is a glycoprotein including at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds.

As used herein, an antibody that “specifically binds to a tumor” refersto an antibody that binds to tumor or tumor antigen. As used herein, anantibody that “specifically binds to a cell” refers to an antibody thatbinds to a cellular antigen. For example the antibody has a K_(D) of5×10⁻⁹ M or less, 2×10⁻⁹ M or less, or 1×10⁻¹⁰ M or less. For example,the antibody is a monoclonal antibody or a polyclonal antibody. Theterms “monoclonal antibody” or “monoclonal antibody composition” as usedherein refer to a preparation of antibody molecules of single molecularcomposition. A monoclonal antibody composition displays a single bindingspecificity and affinity for a target such as cells or a particularcellular epitope. The antibody is for example an IgM, IgE, IgG such asIgG1 or IgG4.

Also useful for systems, method and kits herein is an antibody that is arecombinant antibody. The term “recombinant human antibody”, as usedherein, includes all antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from ananimal (e.g., a mouse). Mammalian host cells for expressing therecombinant antibodies used in the methods herein include for exampleChinese Hamster Ovary (CHO cells) including dhff CHO cells, describedUrlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980 usedwith a DHFR selectable marker, e.g., as described in R. J. Kaufman andP. A. Sharp, 1982 Mol. Biol. 159:601-621, NSO myeloma cells, COS cellsand SP2 cells. In particular, for use with NSO myeloma cells, anotherexpression system is the GS gene expression system shown in WO 87/04462,WO 89/01036 and EP 338,841. To produce antibodies, expression vectorsencoding antibody genes are introduced into mammalian host cells, andthe host cells are cultured for a period of time sufficient to allowexpression of the antibody in the host cells or secretion of theantibody into the culture medium in which the host cells are grown.Antibodies are recovered from the culture medium using standard proteinpurification methods.

Standard assays to evaluate the binding ability of the antibodies towardthe target of various species are known in the art, including forexample, ELISAs, western blots and RIAs. The binding kinetics (e.g.,binding affinity) of the antibodies also can be assessed by standardassays known in the art, such as by Biacore analysis.

General methodologies for antibody production, including criteria to beconsidered when choosing an animal for the production of antisera, aredescribed in Harlow et al. (Antibodies, Cold Spring Harbor Laboratory,pp. 93-117, 1988). For example, an animal of suitable size such asgoats, dogs, sheep, mice, or camels are immunized by administration ofan amount of immunogen, such as the intact protein or a portion thereofcontaining an epitope (e.g., HCC or CD44), effective to produce animmune response. An exemplary protocol is as follows. The animal issubcutaneously injected in the back with 100 micrograms to 100milligrams of antigen, dependent on the size of the animal, followedthree weeks later with an intraperitoneal injection of 100 micrograms to100 milligrams of immunogen with adjuvant dependent on the size of theanimal, for example Freund's complete adjuvant. Additionalintraperitoneal injections every two weeks with adjuvant, for exampleFreund's incomplete adjuvant, are administered until a suitable titer ofantibody in the animal's blood is achieved. Exemplary titers include atiter of at least about 1:5000 or a titer of 1:100,000 or more, i.e.,the dilution having a detectable activity. The antibodies are purified,for example, by affinity purification on columns containing a cellularantigen used for immunization.

The technique of in vitro immunization of human lymphocytes is used togenerate monoclonal antibodies. Techniques for in vitro immunization ofhuman lymphocytes are well known to those skilled in the art. See, e.g.,Inai, et al., Histochemistry, 99(5):335 362, May 1993; Mulder, et al.,Hum. Immunol., 36(3):186 192, 1993; Harada, et al., J. Oral Pathol.Med., 22(4):145 152, 1993; Stauber, et al., J. Immunol. Methods,161(2):157 168, 1993; and Venkateswaran, et al., Hybridoma, 11(6) 729739, 1992. These techniques can be used to produce antigen-reactivemonoclonal antibodies, including antigen-specific IgG, and IgMmonoclonal antibodies.

Pharmaceutical Compositions

An aspect of the present invention provides pharmaceutical compositionsthat include a nanoparticle having at least one of a polymer layer and abinding agent. In certain embodiments, the composition comprises aplurality of nanoparticles that are administered to cells, a tissue, ora subject. The nanoparticles in certain embodiments undergo cellularuptake by for example phagocytosis and endocytosis. In variousembodiments, the nanoparticles are conjugated to a binding agent thatbinds to a molecular antigen of cell or a tissue. In relatedembodiments, the pharmaceutical composition is formulated sufficientlypure for administration to a subject, e.g., a human, a mouse, a rat, adog, a cat, and a cow. The pharmaceutical composition is administeredfor example to an abdomen or a vascular system.

In certain embodiments, the pharmaceutical composition further includesat least one therapeutic agent selected from the group consisting ofgrowth factors, anti-inflammatory agents, vasopressor agents includingbut not limited to nitric oxide and calcium channel blockers,collagenase inhibitors, topical steroids, matrix metalloproteinaseinhibitors, ascorbates, angiotensin II, angiotensin III, calreticulin,tetracyclines, fibronectin, collagen, thrombospondin, transforminggrowth factors (TGF), keratinocyte growth factor (KGF), fibroblastgrowth factor (FGF), insulin-like growth factors (IGFs), IGF bindingproteins (IGFBPs), epidermal growth factor (EGF), platelet derivedgrowth factor (PDGF), neu differentiation factor (NDF), hepatocytegrowth factor (HGF), vascular endothelial growth factor (VEGF),heparin-binding EGF (HBEGF), thrombospondins, von Willebrand Factor-C,heparin and heparin sulfates, and hyaluronic acid. See Toole et al. U.S.Pat. No. 5,902,795 issued May 11, 1999, which is incorporated byreference herein in its entirety.

The therapeutic agent in various embodiments includes an anti-cancer oranti-tumor agent selected from the group of: alkylating agents, such asmechlorethamine, cyclophosphamide, melphalan, uracil mustard,chlorambucil, busulfan, carmustine, lomustine, semustine,streptozoticin, and decrabazine; antimetabolites, such as methotrexate,fluorouracil, fluorodeoxyuridine, cytarabine, azarabine, idoxuridine,mercaptopurine, azathioprine, thioguanine, and adenine arabinoside;natural product derivatives, such as irinotecan hydrochloride,vinblastine, vincristine, dactinomycin, daunorubicin, doxorubicin,mithramycin, taxanes (e.g., paclitaxel) bleomycin, etoposide,teniposide, and mitomycin C; and miscellaneous agents, such ashydroxyurea, procarbezine, mititane, and cisplatinum. See Brown et al.U.S. publication number 20050267069 published Dec. 1, 2005, which isincorporated by reference herein in its entirety.

In other embodiments, the therapeutic agent is a compound, composition,biological or the like that potentiates, stabilizes or synergizes theeffects of another molecule or compound on a cell or tissue. In someembodiments, the drug includes without limitation anti-tumor, antiviral,antibacterial, anti-mycobacterial, anti-fungal, anti-proliferative oranti-apoptotic agents. Drugs that are included in the compositions ofthe invention are well known in the art. See for example, Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman, etal., eds., McGraw-Hill, 1996; and Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 12th Ed., Hardman, et al., eds., McGraw-Hill,2010, the contents of which are herein incorporated by reference herein.

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences 20^(th) Edition by Gennaro, Mack Publishing,Easton, Pa., 2003 provides various carriers used in formulatingpharmaceutical compositions and known techniques for the preparationthereof. Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, sugars such asglucose and sucrose; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil, and soybean oil; glycols such a propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, preservatives andantioxidants can also be present in the composition, the choice ofagents and non-irritating concentrations to be detelmined according tothe judgment of the formulator.

Therapeutically Effective Dose

Methods provided herein involves administering a pharmaceuticalcomposition to cells or to a subject, for example, administering atherapeutically effective amount of a pharmaceutical composition havingnanoparticles having at least one of a polymer layer and a bindingagent. The pharmaceutical composition in certain embodiments optionallyfurther includes a therapeutic agent in such amounts and for such timeas is necessary to achieve the desired result.

The compositions, according to the method of the present invention, areadministered using any amount and any route of administration effectivefor contacting cells or a subject. The exact dosage is chosen by theindividual physician in view of the patient to be treated. Dosage andadministration are adjusted to provide sufficient levels of the activeagent(s) or to maintain the desired effect. Additional factors which maybe taken into account include the severity of the disease state, e.g.,intermediate or advanced stage of a disease condition; age, weight andgender of the patient; diet, time and frequency of administration; routeof administration; drug combinations; reaction sensitivities; andtolerance/response to therapy. Long acting pharmaceutical compositionsmight be administered hourly, twice hourly, every three to four hours,daily, twice daily, every three to four days, every week, or once everytwo weeks depending on half-life and clearance rate of the particularcomposition.

The active agents of the invention are preferably formulated in dosageunit form for ease of administration and uniformity of dosage. Theexpression “dosage unit form” as used herein refers to a physicallydiscrete unit of active agent appropriate for the patient to bediagnosed or to be treated. It will be understood, however, that thetotal daily usage of the compositions of the present invention will bedecided by the attending physician within the scope of sound medicaljudgment. For any active agent, the therapeutically effective dose canbe estimated initially either in cell culture assays or in animalmodels, as provided herein, usually mice, but also potentially fromrats, rabbits, dogs, or pigs. The animal cell model provided herein isalso used to achieve a desirable concentration and total dosing rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of active agentthat can be clearly imaged for diagnosis of cells or tissues (e.g.,tumors) at a very early stage, or that ameliorates the symptoms orprevents progression of a pathology or condition. Therapeutic efficacyand toxicity of active agents can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED50 (the dose is therapeutically effective in 50% of thepopulation) and LD50 (the dose is lethal to 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit can be expressed as the ratio, LD50/ED50. Pharmaceutical compositionswhich exhibit large therapeutic indices are preferred. The data obtainedfrom cell culture assays and animal studies are used in formulating arange of dosage for human use.

Administration of Pharmaceutical Compositions

As formulated with an appropriate pharmaceutically acceptable carrier ina desired dosage, the pharmaceutical composition provided herein isadministered to humans and other mammals topically such as ocularly (asby solutions, ointments, or drops), nasally, bucally, orally, rectally,topically, parenterally, intracisternally, intravaginally, orintraperitoneally.

Injections include intravenous injection, direct or parental injectioninto the tissues (e.g., cancerous and non-cancerous), or injection intothe external layers of the tissue or adjacent tissues, such as forexample injection into the peritoneal cavity, stomach, liver, breast,leg, or lung.

The pharmaceutical composition in various embodiments is administeredwith inert diluents commonly used in the art such as, for example, wateror other solvents, solubilizing agents and emulsifiers such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the delivered compositions canalso include adjuvants such as wetting agents, and emulsifying andsuspending agents.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The activeagent is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives or buffers as may berequired. For example, ocular or cutaneous routes of administration areachieved with aqueous drops, a mist, an emulsion, or a cream.Administration may be diagnostic, prognostic, therapeutic or it may beprophylactic. The invention includes delivery devices, surgical devices,audiological devices or products which contain disclosed compositions(e.g., gauze bandages or strips), and methods of making or using suchdevices or products. These devices may be coated with, impregnated with,bonded to or otherwise treated with a composition as described herein.

Transdermal patches have the added advantage of providing controlleddelivery of the active ingredients to the body. Such dosage forms can bemade by dissolving or dispensing the compound in the proper medium.Absorption enhancers can also be used to increase the flux of thecompound across the skin. The rate can be controlled by either providinga rate controlling membrane or by dispersing the compound in a polymermatrix or gel.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For the purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Theinjectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use. In order to prolong the effect of an active agent, it is oftendesirable to slow the absorption of the agent from subcutaneous orintramuscular injection. Delayed absorption of a parenterallyadministered active agent may be accomplished by dissolving orsuspending the agent in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the agent in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofactive agent to polymer and the nature of the particular polymeremployed, the rate of active agent release can be controlled. Examplesof other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the agent in liposomes or microemulsions which are compatiblewith body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the active agent(s) of theinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active agent(s).

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeagent is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, 0 absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as milksugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings, release controlling coatings and other coatings well known inthe pharmaceutical formulating art. In such solid dosage forms theactive agent(s) may be admixed with at least one inert diluent such assucrose or starch. Such dosage forms may also comprise, as is normalpractice, additional substances other than inert diluents, e.g.,tableting lubricants and other tableting aids such a magnesium stearateand microcrystalline cellulose. In the case of capsules, tablets andpills, the dosage forms may also comprise buffering agents. They mayoptionally contain opacifying agents and can also be of a compositionthat they release the active agent(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes.

A portion of the invention is provided in a publication in the JournalNano Letters and is entitled “Nanomaterials for X-ray Imaging: GoldNanoparticle-Enhancement of X-ray Scatter Imaging of HepatocellularCarcinoma” by Danielle Rand, Vivian Ortiz, Yanan Liu, Zoltan Derdak,Jack R. Wands, Milan Tati{hacek over (c)}ek, and Christoph Rose-Petruck(Rand et al. 2011 Nano Lett. 11: 2678-2683), which is herebyincorporated by reference in its entirety.

The following examples and claims are illustrative only and not intendedto be further limiting. Those skilled in the art will recognize or beable to ascertain using no more than routine experimentation, numerousequivalents to the specific procedures described herein. Suchequivalents are within the scope of the present invention and claims.The contents of all references including issued patents and publishedpatent applications cited in this application are hereby incorporated byreference in their entireties. A skilled person will recognize that manysuitable variations of the methods may be substituted for or used inaddition to those described above and in the claims. It should beunderstood that the implementation of other variations and modificationsof the embodiments of the invention and its various aspects will beapparent to one skilled in the art, and that the invention is notlimited by the specific embodiments described herein and in the claims.Therefore, it is contemplated to cover the present embodiments of theinvention and any and all modifications, variations, or equivalents thatfall within the true spirit and scope of the basic underlying principlesdisclosed and claimed herein.

EXAMPLES Example 1 Materials

Chemicals were purchased from Sigma-Aldrich Inc. (St. Louis, Mo.) unlessotherwise specified. Uncoated gold nanoparticles (ten nm and 50 nm)stabilized in citrate buffer were purchased from British BiocellInternational (Cardiff, UK): 10 nm gold nanoparticles were BBI catalog#EMGC10, batch #15683; and 50 nm gold nanoparticles were BBI catalog#EMGC50, batch #15693.

Example 2 Layer-by-Layer Coating of Nanoparticles

Stock solutions of poly(acrylic acid) (PAA) and poly(allylaminehydrochloride) (PAH) were prepared (10 mg/mL PAA or PAH in onemillimolar (mM) aqueous sodium chloride solution). Unpurified onemilliliter (mL) aliquots of ten nm gold nanoparticles or of 50 nm goldnanoparticles were mixed with 100 microliters (μL) of 1 mM sodiumchloride and 200 μL of PAA stock solution. The mixture was incubated for30 minutes. Excess polymer in the supernatant was removed bycentrifugation. Anionic poly(acrylic acid) (PAA) was deposited on thegold nanoparticles, resulting in a gold nanoparticle being coated withcarboxylic acid groups located on the nanoparticle surface (FIG. 1;AU-PAA nanoparticle).

The PAA-coated nanoparticle were re-suspended in phosphate bufferedsaline (PBS). The PAA encapsulated gold nanoparticles (one mL aliquots)were then mixed with 100 μL of 1 mM sodium chloride and 200 μL ofcationic poly(allylamine hydrochloride) stock solution for 30 minutes.The electrostatic interaction between the oppositely charged PAA and PAHpolymer layers yielded a layer-by-layer coating of the PAA and PAHpolyelectrolytes on the surface of the nanoparticles.

The mixture was then centrifuged to remove excess polymer in thesupernatant. The resulting gold nanoparticles were coated with an innerlayer of PAA polymer and an outer layer of PAH polymer. Solutionscontaining the PAA-PAH coated gold nanoparticles were stored at roomtemperature.

Example 3 Preparation of FOCUS Cells

FOCUS cells were cultured and maintained at 37° C. (5% CO₂) in Eagle'sMinimum Essential Medium (EMEM) supplemented with 10% fetal bovineserum, 1% penicillin/streptomycin and 1% L-glutamine. Cells were grownto confluence and formed a monolayer. Trypsin was added to detach theFOCUS cells, which were re-suspended in serum-free EMEM.

Example 4 Incubation of FOCUS Cells with Coated Nanoparticles

To prepare samples for X-ray scatter imaging, cell pellet samplescontaining approximately 10⁷ FOCUS cells were incubated in vials withnanoparticle solutions containing either ten nm PAA-PAH coated goldnanoparticles, or 50 nm PAA-PAH coated gold nanoparticles. Control cellpellet samples were incubated with either no gold, or with ten nmuncoated gold nanoparticles only, viz., no PAA-PAH coating. The numberof FOCUS cells in the cell pellet samples was chosen to model the sizeof a small tumor having a diameter of a few millimeters.

After each incubation, the cells were collected, washed and imaged. Theapproximate amount of gold nanoparticles contained in each incubationwith the cell pellet samples was measured in Example 8 by spectrometricmethods.

Example 5 Spatial Frequency Heterodyne Imagine of the FOCUS Cells andNanoparticles

Spatial frequency heterodyne imaging was used to image samples of FOCUScell pellets contacted with gold nanoparticles. The spatial frequencyheterodyne imaging system shown in FIG. 2 directed X-ray radiation to anabsorption grid and a sample, and then detected the X-rays scattered bythe sample to using a detector and Fourier transformation (Stein, A. F.et al. 2010 Opt. Express 18: 13271-13278; Wen, H. et al. 2009 Radiology251: 910-918; and Wen, H. et al. 2008 IEEE Trans Med Imaging 27:997-1002). A vial holder positioned the vials containing the cellpellets-gold nanoparticles.

The X-ray scatter measurements were obtained with a micro-focus X-raytube (Trufocus Corporation, model: TFX-3110EW) with a tungsten anode.The tube was operated at an electrical power of 20 watts (W), producinga maximum voltage of 95.6 kilovolts (kV). High voltages reduced theexposure times, and were observed to be better suited for in vivoapplications, as imaging a tissue in a subject required largepenetration depths. The distance between the source and camera was 1.6meters (m), and the sample was placed at a distance halfway (0.8 m)between the source and camera to enhance resolution (Wen, H. et al. 2009Radiology 251: 910-918).

The absorption grid was stainless steel with a pitch of 50 micrometer(μm), and was purchased from Small Parts, Inc. (Seattle, Wash.). Thegrid was positioned directly in front of the vials containing the cellpellet samples and the gold nanoparticles. The images were acquired withan X-ray CCD camera (Princeton Instruments, Model Quad-RO 4096). Thetotal exposure time for each image was 180 seconds.

Example 6 Fourier Transformation of Images

Fourier transformation was then performed on the original images (FIG. 3bottom left image; F, Original). The Fourier transformation convertedthe product of X-ray scatter transmittances of the sample and the gridinto a convolution in the spatial frequency domain (see FIG. 3). Thegrid, a periodic structure, produced a series of peaks in thisconvolution. Each peak produced by the grid was “surrounded” by thespatial frequency spectrum of the sample.

Selecting an area around a specific peak in the convolution and Fourierback-transforming this area yielded a logarithm of the scatteredintensities to real space and provided a processed image that containedanisotropic information about the scattering of the incident X-rays bythe sample. The area surrounding the central 0^(th)-order peak (FIG. 3bottom right image; F⁻¹, S₀: 0^(th) order) corresponded to the originalX-ray absorption image without scatter, which was then used fornormalizing the images. The X-ray absorption image without scatter wassubtracted from the higher order images to remove absorption featuresthat distort clear visualization of the cell pellet samples.

The area around the 1^(st)-order peak to the immediate left of the0^(th)-order peak (FIG. 3 bottom center image; F⁻¹, S₁: 1^(st) order,left) corresponded to scattering in the x-direction, and thereforeproduced a processed “left 1^(st)-order” scatter image upon Fourierback-transformation and normalization. Further, the area around the1^(st)-order peak immediately above the 0^(th)-order peak (FIG. 3 topimage top right box) corresponded to scattering in the y-direction, andtherefore producing a processed “upper 1^(st)-order” scatter image uponFourier back-transformation and normalization. Thus, the original X-rayimage obtained yielded two Fourier transformation processed images. Aprocessed image resulting from X-radiation scattered horizontally (FIG.3 bottom center image; F⁻¹, S₁: 1^(st) order), and the processed imagefrom x-radiation scattered vertically (FIG. 3 top image top right box).Both of these 1^(st) order images produced identical scatter signalsbecause of the isotropic scattering of the spherical nanoparticles.

Example 7 Calculation of Scatter Signals and Enhancement Factors

The scattering signal (S) for each processed image was calculatedaccording to the following equation:

$S = {- {\log \left( \frac{I_{1}/G_{1}}{I_{0}/G_{0}} \right)}}$

such that I₁ and I₀ were the detected X-ray signals with sample in the1^(st) and 0^(th) order, respectively. In the equation above, G₁ and G₀were the detected X-ray signals without sample in the 1^(st) and 0^(th)order, respectively. The signal S was calculated for each vial in thearea that contains the cell pellet (S_(cells)) and the area of thesupernatant (S_(super)). Precautions were taken to avoid detecting anysignal from the walls of the vials. The normalized scattering signal(S_(norm)) for each cell pellet was calculated by dividing the signalmeasured in the area containing the cell pellet (S_(cells)) by thesignal measured in the area of the supernatant (S_(super)) as shown inthe equation below:

$S_{norm} = \frac{S_{cells}}{S_{super}}$

The normalized scattering signal was then compared to the absorbancemeasured in the original absorption X-ray images. The absorbance wascalculated using the following equation:

$A = {- {\log \left( \frac{I_{sample}}{I_{{flat}\mspace{14mu} {field}}} \right)}}$

such that, I_(sample) was the detected X-ray intensity with a samplevial and I_(flat field) was the detected X-ray intensity without asample vial. The absorbance was calculated for each vial in the areacontaining the cell pellet (A_(cells)) and in the area of thesupernatant (A_(super)), respectively. The normalized absorbance signal(A_(norm)) measured for each pellet in the absorption images wascalculated by dividing the signal measured in the area containing thecell pellet (A_(cells)) by the signal measured in the area of thesupernatant (A_(super)) using the equation shown below:

$A_{norm} = \frac{A_{cells}}{A_{super}}$

The scattering signal (S_(norm)) was compared to the absorption signal(A_(norm)) measured in the absorption images by calculating anenhancement factor as shown in an equation below:

${{Enhancement}\mspace{14mu} {factor}} = \frac{S_{norm}}{A_{norm}}$

Example 8 ICP-AES Analysis of Gold in Cell Pellet Samples

The signal enhancement in the processed X-ray images due to increasedscattering by the gold nanoparticles was normalized byspectrophotometric techniques for gold content (mass) in the cells.After the pellets were imaged using X-ray scattering, the amount of goldtaken up by the FOCUS cell pellets during incubation was determinedusing inductively-coupled plasma atomic emission spectroscopy (ICP-AES).

Samples were prepared for ICP-AES analysis by digesting gold andorganics with aqua regia (1:3 mixture of HNO₃:HCl) followed by dilutionin 2% nitric acid. Data from ICP-AES analysis showed that the samplescontained four micrograms to eight micrograms (μg), corresponding toseveral hundred 50 nm nanoparticles per cell and tens of thousands often nm nanoparticles per cell (shown in Tables 1 and 2).

Example 9 Images Analysis

FIG. 4 panels A-D are representative photographs (FIG. 4 panel A),absorption images (FIG. 4 panel B) and X-ray scatter images (FIG. 4panels C-D) of vials containing FOCUS cell pellets incubated with either50 nm PAA-PAH coated gold nanoparticles (FIG. 4 left image in eachpanel), or ten nm. PAA-PAH coated gold nanoparticles (FIG. 4 right imagein each panel). Control samples of FOCUS cell pellets were incubatedwith no gold nanoparticles (FIG. 4 center image in each panel).

Visual analysis of photographs clearly indicated that samples incubatedwith PAA-PAH coated gold nanoparticles were labeled with gold, and thatcontrol samples incubated with no gold nanoparticles were not labeled(FIG. 4 panel A). Cell pellets containing gold nanoparticles werestained a distinctive brown color. It was observed that for each of thesamples the EMEM supernatant located above the cell pellet was naturallypink and showed no presence of gold nanoparticles. No clear labeling ofFOCUS cell pellets was observed in images obtained using conventionalabsorption techniques. For the absorption images, FOCUS cell pelletsincubated with gold nanoparticles and the cell pellet samples incubatedwith no gold nanoparticles were indistinguishable from each other (FIG.4 panel B). Most important, images obtained by spatial frequencyheterodyne imaging showed a signal enhancement in samples incubated withthe PAA-PAH coated gold nanoparticles compared to control cells havingno nanoparticles because of uptake of the gold nanoparticles into thecell pellets (FIG. 4 panels C-D).

The signal enhancement for spatial frequency heterodyne imaging comparedto absorbance imaging was calculated by first obtaining an averageintensity profile of the absorbance image at the pellet height(A_(cells)), such that the average absorbance intensity varied dependingon presence of gold nanoparticle concentration contacting the cells. Theabsorbance value was normalized using the intensity of the supernatant(A_(super)) which was approximately the same for each of the samplesirrespective of whether samples contained gold nanoparticles or not. Theupper right boxes and two lower boxes shown in FIG. 4 panels B-D outlinethe areas selected for calculating intensity profiles of the supernatantand pellet, respectively. The intensity values were averaged using theoptical data from the outlined areas. Examples herein used a vial holderfor positioning the vials containing the cell pellets-gold nanoparticlesdescribed above ensured that the average signal intensities werestandardized for each image obtained, as absorbance and signalcomparisons between the samples were obtained from the same relativepositions (having the same thickness) in the vials containing thesamples.

Data show that gold nanoparticles not coated with PAA and PAHpolyelectrolytes were not effectively uptaken by the FOCUS cell pellets.Data show that less than 25% of the uncoated nanoparticles used forincubation were phagocytosed by the FOCUS cells after the one hourcontact with the nanoparticles. Clearly these control cell pellets wereobserved to have reduced intensity due to the relatively low goldconcentrations in each cell (see Table 1).

Coating gold nanoparticles with the PAA-PAH polymer coatings enhancedFOCUS cell pellets cellular uptake (e.g., phagocytosis) of thenanoparticles compared using non-coated control nanoparticles.Layer-by-layer coating of the nanoparticles resulted in increased amountof gold in each cell, and therefore the increased scattering signalobserved in the X-ray images obtained (Table 1). Data in Table 1 furthershow that the FOCUS cells phagocytosed more than twice as many ten nmPAA-PAH coated gold nanoparticles (275,000 nanoparticles) as ten nmuncoated gold nanoparticles (108,000 nanoparticles).

Most important, FOCUS cells phagocytosed twice as much mass of gold percell of 10 nm PAA-PAH coated gold nanoparticles (2.8±0.4 picograms percell) and 50 nm PAA-PAH coated gold nanoparticles (2.2±0.3 picograms percell) compared to uncoated gold nanoparticles (1.2±0.5 picograms percell). The uptake of the nanoparticles was enhanced by the PAA-PAHcoating, as data showed that FOCUS cells phagocytosed an average ofapproximately 275,000 polyelectrolyte-coated ten nm PAA-PAH goldnanoparticles per cell, compared to approximately 108,000 uncoated tennm gold nanoparticles per cell (Table 1).

It was observed that FOCUS cell pellets phagocytosed approximately thesame amount of PAA-PAH coated nanoparticles irrespective if thenanoparticles were ten nm (2.8±0.4 picograms) or 50 nm (2.2±0.3) in size(Table 1). Without being limited by any particular theory or mechanismof action, it is here envisioned that the total volume of goldnanoparticles incubated with the cells that is an important factor indetermining the extent of cellular uptake. Calculation of the percentagevolume of the nanoparticles in the cell indicated than only a very smallportion of each cell is actually occupied by the coated goldnanoparticles. Data show that the FOCUS cells phagocytosed a cell volumeof much less than 0.001% of PAA-PAH polyelectrolyte-coated nm goldnanoparticles: 10 nm PAA-PAH coated gold nanoparticles (0.00063%), 50 nmPAA-PAH coated gold nanoparticles (0.00049%), and uncoated 10 nm goldnanoparticles (0.00025%).

Analysis of the absorption X-ray images further shows that the signalmeasured for FOCUS cell pellets incubated with PAA-PAH coated goldnanoparticles was 1.2% greater than the signal measured for FOCUS cellpellets incubated without gold nanoparticles (see Table 2). However, ofthe total number (12) of absorption images taken of FOCUS cell pelletslabeled with gold nanoparticles, only 58% (seven) were observed to haveexhibit any enhancement due to gold labeling by the nanoparticles. Thelarge standard deviation calculated for absorption techniquesillustrates the extent that absorption images are unreliable for imagingcells in vitro, let alone in vivo.

The spatial frequency heterodyne imaging described herein visualized anddifferentiated between FOCUS cell pellets incubated with PAA-PAH coatedgold nanoparticles and control FOCUS cell pellets incubated with no goldnanoparticles. The left 1^(st)-order scatter images (FIG. 4 panel C) andupper 1^(st)-order scatter images (FIG. 4 panel D) produced usingspatial frequency heterodyne imaging were analyzed and used to producenormalized intensity profiles for the cell pellet samples. Thesecalculated normalized profiles corresponded to the logarithm of theintensity of the scattered x-radiation. Without being limited by anyparticular theory or mechanism of action, it is envisioned that thesignal differences between the 1^(st) order images were due to the sideinterfaces produced by either air in the vials (FIG. 4 panel C) or thebottom interface of the vials (FIG. 4 panel D). Thus the signaldifferences in the 1^(st)-order spatial frequency heterodyne imagingwere from anisotropic X-ray scattering at smooth material interfaces.

Analysis of processed spatial frequency heterodyne (X-ray scatter)images and the ICP-AES data in Examples herein shows an average signalenhancement due to gold labeling that ranges from approximately 1.6% to4.4% (Table 2). Of the X-ray scatter images (24 total images) taken ofFOCUS cell pellets labeled with gold nanoparticles, 88% (21 images)showed a signal enhancement when compared to X-ray scatter images ofFOCUS pellets containing no gold. Overall, the data herein of processedspatial frequency heterodyne images and ICP-AES show an at least threeor at least a five enhancement factor per picogram of gold uptaken bythe cell (Table 2).

These data show that for every picogram of gold taken up by a cell, thesignal observed in the processed scatter images was enhanced by anaverage of 3.6% and 5.7% (Table 2; replicate 1 and replicate 2,respectively). It was observed that each cell contained an average ofabout three picograms of polyelectrolyte-coated gold nanoparticles(Table 1), resulting in a potential signal enhancement due to goldlabeling of approximately 11% to approximately 17% in logarithm scale(Table 2).

Example 10 Model of In Vivo Imaging Using Nanoparticles and SpatialFrequency Heterodyne Imaging

Examples herein show development of an X-ray scatter imaging system forin vivo imaging of cells and tissues. The in vivo model utilized signalenhancement produced by gold nanoparticle labeling such that the signalenhancement would correspond to visualizing cells and structures beneathmany layers of tissue.

Spatial frequency heterodyne imaging was performed as described in FIG.2 for a cell pellet sample incubated with 50 nm PAA-PAH coated goldnanoparticles, in which X-ray radiation progressed through onecentimeter of water prior to reaching the cell pellets previouslycontacted with nanoparticles. X-ray radiation traversing one centimeterof water positioned above the cell pellet-gold nanoparticles was chosento imitate closely a thickness of tissue which X-ray radiation deliveredto a patient's skin might traverse at the point of entry into thepatient.

Water and liver tissues have similar radiological densities, in fact theelectron densities of water and liver tissues differ by less than 5%(Yang, M. et al. 2010 Physics in Medicine and Biology 55: 1343-1362).X-ray scatter imaging an additional traversal through one centimeter ofwater resulted in the detection of gold-labeled FOCUS cell pellets. Datashow that normalized signal intensities in the scatter images increasedby 1.8±0.5% for cells incubated with gold nanoparticles compared to acontrol cell pellet incubated with no gold nanoparticles.

Example 11 Imaging Cells Using Antibody-Conjugated Nanoparticles

Nanoparticles linked to HCC-specific antibodies were prepared todetermine whether such compositions would be useful as X-ray visibleimmunolabels, for in vivo detection and X-ray imaging of HCC tumors in amurine mouse model.

The FB50 monoclonal antibody was generated by cellular immunization ofmice with FOCUS HCC cells. The FB50 antibody specifically binds andtargets FOCUS cells, and the antibody is localized intracellularly. Toreduce nonspecific uptake of the gold nanoparticles by the other typesof tissue, the nanoparticle surface was coated with a polyethyleneglycol (PEG). Without being limited by any particular theory ormechanism of action, it is here envisioned that PEG prevented thenon-specific adsorption of the proteins onto the nanoparticle surface,allowing for longer circulation times in vivo and enhanced accumulationof the nanoparticle specifically in the liver of the subject.

Bi-functional PEG (HS-PEG-COOH) was added to the nanoparticle, forfacile attachment to the nanoparticle surface. Bio-conjugation of thegold nanoparticles to the FB50 antibody was performed using techniquesinvolving EDC/NHS cross-linking chemistry. The bio-conjugation involvedlinking the carboxylic acid groups on the PEG coating around thenanoparticles to amine groups present on the FB50 antibody (See FIG. 5).Dynamic light scattering techniques showed that each of the steps ofproducing the PEG coated the gold nanoparticles and the conjugated FB50antibody resulted in increases to the diameter of the 50 nm goldnanoparticles (FIG. 6 panel A). Specifically data showed that theuncoated 50 nm gold particles had a diameter of 71.3±10.0 nm, that thePEG-coated 50 nm gold nanoparticles had a diameter of 81.8±12.1 nm, andthat the PEG-coated FB50 antibody conjugated 50 nm gold nanoparticleshad a diameter of 93.9±18.3 nm (FIG. 6 panel A). Without being limitedby any particular theory or mechanism of action, it is here envisionedthat PEG coated on the nanoparticle surface prevented the non-specificadsorption of the antibody-conjugated nanoparticles to cells notexpressing HCC, allowing for longer circulation times in vivo andenhanced accumulation of the nanoparticle specifically in the liver ofthe subject.

Cellular uptake by FOCUS cells pellets and by NIH/3T3 cell pellets ofthe PEG coated gold nanoparticles or the PEG coated FB50 conjugated tennm gold nanoparticles was measured (FIG. 6 panel B). The specificity ofFB50 antibody was evaluated by using as a negative control the NIH/3T3fibroblasts, which do not express HCC. An anti-Murutucu tropical virus(MUK) antibody was also used as an additional negative control antibodyfor the FOCUS cells, as the MUK antibody does not specifically bind HCC(FIG. 6 panel B).

Data show that at least thirty fold more of the PEG-coated FB50 antibodyconjugated ten nm gold nanoparticles were taken into the FOCUS cellpellets compared to the NIH/3T3 cell pellets. Further, little or noPEG-coated MUK antibody conjugated gold nanoparticles were transportedinto the FOCUS cell or the NIH/3T3 cells. Thus, the PEG-coated FB50antibody conjugated ten nm gold nanoparticles specifically bound to theHCC antigen and enhanced cellular uptake of the nanoparticles into theFOCUS cells.

Example 12 In Vivo Imaging of Livers

Examples herein used the PEG/FB50 antibody conjugated nanoparticles weretested in an in vivo a murine mouse model. Mice were injected twice overa 24 hour period into the tail vein with 50 nm PEG/FB50 antibodyconjugated gold nanoparticles, or saline as a negative control. Subjectswere sacrificed 48 hours after the first injection, and were fixed informaldehyde and imaged in the spatial frequency heterodyne imagingsystem (FIG. 2) and imaging was performed in situ. Without being limitedby any particular theory or mechanism of action, it is envisioned thatinjected nanoparticles that were not conjugated to the targeting/bindingagent FB50 antibody such as the Au-PEG nanoparticles (shown in FIG. 4)concentrated mainly in the liver due to the high phagocytic activity ofthe Kuppfer cells located in that tissue. In vivo spatial frequencyheterodyne imaging was performed in the area of the liver of eachsubject.

In situ imaging showed that 80% of livers in subjects injected withPEG/FB50 antibody conjugated gold nanoparticles (FIG. 7 panel A rightcolumn bottom row) exhibited a brighter normalized scatter signal thanlivers from subjects injected with the saline control (FIG. 7 panel Aright column top row).

Livers were excised from subjects and spatial frequency heterodyneimaging system was performed. It was observed that subjects injectedwith PEG/FB50 antibody conjugated gold nanoparticles also clearly showedenhanced signal enhancement of the liver compared to spatial frequencyheterodyne images of livers from control subjects injected with salineonly (FIG. 7 panel B). The spatial frequency heterodyne imaging was alsoperformed in situ for spleens, kidneys and lungs from subjects, and theliver was the primary organ to show significant signal enhancement withan average enhancement of 23.0±14.1%. Enhanced signal enhancements wereobserved in both absorption images (FIG. 7 panel B left images) andspatial frequency heterodyne imaging (FIG. 7 panel B right images). Agreater than a factor of ten signal enhancement was calculated in thescatter images compared to the absorption images. Thus, spatialfrequency heterodyne imaging effectively visualized in vivo the size andcontours of in vivo organs and greatly outperformed the results obtainedby absorption imaging.

Example 13 In Vivo Imaging of Tissues in the Body

Nanoparticles (metal nanoparticles, metal oxide nanoparticles, and MRIagent nanoparticles) are prepared for in vivo administering and imagingof tissues including muscle, bone, cartilage, skin, and blood vessels.

The nanoparticles are contacted with a polymer such that a polymer layercoated the nanoparticles. The polymer layer provides the nanoparticleswith a hydrophobic barrier that enhances non-specific cellular uptake.Alternatively, nanoparticles are constructed with both a polymer and abinding agent such that the binding agent extends from the nanoparticleto specifically bind to molecular antigens present on and in cells andtissues.

The nanoparticles described herein are stable at a range of storagetemperatures (e.g., room temperature and below freezing) and arenon-toxic to subjects. The nanoparticles are formulated intocompositions and injected into subjects for imaging.

Absorption imaging and spatial frequency heterodyne imaging areperformed in vivo by directing X-ray radiation to tissues of subjectsinjected with nanoparticles. Deflection of incident X-rays from theprimary beam direction are detected in Examples herein by placing anabsorption grid between the sample and the X-ray source. Fouriertransformations are performed on the original X-ray scatter images toobtain processed images that analyzed by a blind panel of doctors toallow for histological information of the tissues.

The samples of the tissues are then excised, fixed and/or decalcified.Histopathology slides for each tissue are prepared, and multiple imagesare acquired using a standard microscope and an image analysis software.The panel of doctors then uses the images obtained from the histologicalslides to identify the type and histological condition of each of thetissues. Data analyses are performed (e.g., sensitivity and specificity)to compare the results obtained from each of the absorption imaging,spatial frequency heterodyne imaging, and the actual histologicalanalysis. Data show that spatial frequency heterodyne imaging usingmetal nanoparticles, metal oxide nanoparticles, and MRI agentnanoparticles is much more sensitive in imaging and differentiatingtissue than absorption imaging. Most important, the spatial frequencyheterodyne imaging of tissues injected with the nanoparticles is aseffective in diagnosing tissue conditions as actual histologicalanalysis.

1-13. (canceled)
 14. A method of identifying in a model system apotential therapeutic agent for treating or preventing a diseasecondition, the method comprising: contacting a first sample and a secondsample of cells or tissue having the disease condition with acomposition including: a nanoparticle and at least one of a polymerlayer coating the nanoparticle and a binding agent that specificallybinds the disease agent; contacting the second sample with the potentialtherapeutic agent; and, measuring a presence or an amount of a marker inthe first sample and the second sample, wherein the marker ischaracteristic of the disease condition, wherein a greater amount of themarker in the first sample compared to that in the second sample is ameasure of treatment and protection by the potential therapeutic agent,thereby identifying the potential therapeutic agent for treating orpreventing the disease condition.
 15. The method according to claim 14,wherein detecting the presence of the marker in the first sample and thesecond sample comprises measuring or detecting the nanoparticle usingX-ray scatter imaging or spatial frequency heterodyne imaging.
 16. Themethod according to claim 14, wherein prior to contacting, the methodfurther comprises constructing the nanoparticle or a plurality ofnanoparticles comprising at least one material selected from the groupof: a metal, a metal oxide, a magnetic resonance imaging agent, and acombination thereof.
 17. The method according to claim 16, whereinconstructing the nanoparticle comprises forming a shell or core of thenanoparticle with at least one from the group of: with at least one of:silver, copper, gold, cadmium, zinc, nickel, palladium, platinum,rhodium, platinum, manganese, gadolinium, dysprosium, tantalum,titanium, and iron.
 18. The method according to claim 14, wherein thebinding agent comprises at least one selected from the group of: a drug,a protein, a carbohydrate, and a nucleotide sequence.
 19. The methodaccording to claim 14, wherein the disease condition is associated withor produced by at least one from the group of: a virus, a tumor, acancer, a fungus, a bacterium, a parasite, a pathogenic molecule, and aprotein.
 20. The method according to claim 14, wherein prior tocontacting, the method further comprises constructing the nanoparticleby attaching or conjugating the binding agent to an external surface ofthe nanoparticle, wherein the binding agent comprises at least oneselected from the group of: a drug, a protein, a carbohydrate, and anucleotide sequence.
 21. The method according to claim 20, wherein theprotein is an antibody selected from the group of: a monoclonalantibody; a polyclonal antibody; a single-chain antibody (scFv); arecombinant heavy-chain-only antibody (VHH); an Fv; a Fab; a Fab′; and aF(ab′)₂.
 22. The method according to claim 14, wherein measuring furthercomprises observing a localization of the marker in the cells or thetissue of the first sample and the second sample.
 23. The methodaccording to claim 14, further comprising spatial frequency heterodyneimaging the nanoparticles in the cells or the tissue using an absorptiongrid and a detector.
 24. A kit for imaging cells or a tissue in asubject comprising: a composition comprising a nanoparticle including atleast one selected from the group of: a polymer layer and a bindingagent, wherein the composition binds to and/or is phagocytosed by thecells or the tissue; instructions for use, wherein the instructionsdescribe: contacting the cells or the tissue with the composition, andimaging the cells and the tissue and detecting X-ray scattering of thecomposition with a device; and a container.
 25. The kit according toclaim 24, wherein the nanoparticles comprise at least one selected fromthe group of: a metal, a metal oxide, an inorganic material, an organicmaterial, a magnetic resonance imaging agent, and a combination thereof.26. A composition for imaging cells or a tissue comprising: a metalnanoparticle having attached to an external surface of the nanoparticlea polymer layer, and FB50 monoclonal antibody that specifically binds anantigen of hepatocellular carcinoma, wherein the polymer layer comprisesat least one of: a polyethylene glycol, a polyelectrolyte, an anionicpolymer, and a cationic polymer.